Mini-dystrophin nucleic acid sequences

ABSTRACT

The present invention relates to compositions and methods for expressing mini-dystrophin peptides. In particular, the present invention provides compositions comprising nucleic acid sequences that are shorter than wild-type dystrophin cDNA and that express mini-dystrophin peptides that function in a similar manner as wild-type dystrophin proteins. The present invention also provides compositions comprising mini-dystrophin peptides, and methods for expressing mini-dystrophin peptides in target cells.

The present Application is a National State Entry under 35 U.S.C. 371 ofPCT application PCT/US01/31126, filed Oct. 4, 2001, which claimspriority to U.S. Provisional application Ser. No. 60/238,848, filed Oct.6, 2000, both of which are herein incorporated by reference.

This invention was made with Government support under contract NIHR01AR40864-10. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for expressingmini-dystrophin peptides. In particular, the present invention providescompositions comprising nucleic acid sequences that are shorter thanwild-type dystrophin cDNA and that express mini-dystrophin peptides thatfunction in a similar manner as wild-type dystrophin proteins. Thepresent invention also provides compositions comprising mini-dystrophinpeptides, and methods for expressing mini-dystrophin peptides in targetcells.

BACKGROUND OF THE INVENTION

Muscular dystrophy is a group of inherited disorders characterized byprogressive muscle weakness and loss of muscle tissue. Musculardystrophies includes many inherited disorders, including Becker'smuscular dystrophy and Duchenne's muscular dystrophy, which are bothcaused by mutations in the dystrophin gene. Both of the disorders havesimilar symptoms, although Becker's muscular dystrophy is a slowerprogressing form of the disease. Duchenne's muscular dystrophy is arapidly progressive form of muscular dystrophy.

Both disorders are characterized by progressive muscle weakness of thelegs and pelvis which is associated with a loss of muscle mass(wasting). Muscle weakness also occurs in the arms, neck, and otherareas, but not as severely as in the lower half of the body. Calfmuscles initially enlarge (an attempt by the body to compensate for lossof muscle strength), the enlarged muscle tissue is eventually replacedby fat and connective tissue (pseudohypertrophy). Muscle contractionsoccur in the legs and heels, causing inability to use the musclesbecause of shortening of muscle fibers and fibrosis of connectivetissue. Bones develop abnormally, causing skeletal deformities of thechest and other areas. Cardiomyopathy occurs in almost all cases. Mentalretardation may accompany the disorder but it is not inevitable and doesnot worsen as the disorder progresses. The cause of this impairment isunknown. Becker's muscular dystrophy occurs in approximately 3 out of100,000 people. Symptoms usually appear in men between the ages of 7 and26. Women rarely develop symptoms. There is no known cure for Becker'smuscular dystrophy. Treatment is aimed at control of symptoms tomaximize the quality of life. Activity is encouraged. Inactivity (suchas bed rest) can worsen the muscle disease. Physical therapy may behelpful to maintain muscle strength. Orthopedic appliances such asbraces and wheelchairs may improve mobility and self-care. Becker'smuscular dystrophy results in slowly progressive disability. A normallife span is possible; however, death usually occurs after age 40.

Duchenne's muscular dystrophy occurs in approximately 2 out of 10,000people. Symptoms usually appear in males 1 to 6 years old. Females arecarriers of the gene for this disorder but rarely develop symptoms.There is no known cure for Duchenne's muscular dystrophy. Treatment isaimed at control of symptoms to maximize the quality of life. Activityis encouraged. Inactivity (such as bed rest) can worsen the muscledisease. Physical therapy may be helpful to maintain muscle strength andfunction. Orthopedic appliances such as braces and wheelchairs mayimprove mobility and the ability for self-care. Duchenne's musculardystrophy results in rapidly progressive disability. By age 10, bracesmay be required for walking, and by age 12, most patients are confinedto a wheelchair. Bones develop abnormally, causing skeletal deformitiesof the chest and other areas. Muscular weakness and skeletal deformitiescontribute to frequent breathing disorders. Cardiomyopathy occurs inalmost all cases. Intellectual impairment is common but is notinevitable and does not worsen as the disorder progresses. Death usuallyoccurs by age 15, typically from respiratory (lung) disorders.

Although there are no available treatments for muscular dystrophy, theusefulness of gene replacement as therapy for the disease has beenestablished in transgenic mouse models. Unfortunately, progress towardtherapy for human patients has been limited by lack of a suitabletechnique for delivery of such vectors to large masses of muscle cells.What is needed in the art is a vector that can carry most of thedystrophin coding sequence, that can be cheaply produced in largequantities, that can be delivered to a large mass of muscle cells, andthat provides stable expression of dystrophin after delivery.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for expressingmini-dystrophin peptides. In particular, the present invention providescompositions comprising nucleic acid sequences that are shorter thanwild-type dystrophin cDNA and that express mini-dystrophin peptides thatfunction in a similar manner as wild-type dystrophin proteins. Thepresent invention also provides compositions comprising mini-dystrophinpeptides, and methods for expressing mini-dystrophin peptides in targetcells.

The present invention provides such shortened nucleic acid sequences ina variety of ways. For example, the present invention provides nucleicacids encoding only 4, 8, 10, 12, 14, 16, 18, 20 and 22 spectrin-likerepeat encoding sequences (i.e. nucleic acids encoding an exact numberof spectrin-like repeats). As wild-type dystrophin has 24 spectrin-likerepeat encoding sequences, providing nucleic acids encoding fewernumbers of repeats reduces the size of the dystrophin gene (e.g.allowing the nucleic acid sequence to fit into vectors with limitedcloning capacity). Another example of such shortened nucleic acidsequences are those that lack at least a portion of the carboxy-terminaldomain of wild-type dystrophin nucleic acid. A further example of suchshortened nucleic acid sequences are those that lack at least a portionof the 3′ untranslated region, or 5′ untranslated region, or both. Incertain embodiments, the present invention provides compositionscomprising the peptides expressed by the nucleic acid sequences of thepresent invention.

In certain embodiments, the present invention provides compositionscomprising nucleic acid encoding a mini-dystrophin peptide, wherein themini-dystrophin peptide comprises a spectrin-like repeat domain, andwherein the spectrin-like repeat domain consists of n spectrin-likerepeats, wherein n is an even number less than 24. In particularembodiments, the present invention provides nucleic acid encoding amini-dystrophin peptide, wherein the mini-dystrophin peptide comprises aspectrin-like repeat domain comprising n spectrin-like repeats, whereinthe mini-dystrophin peptide contains no more than n spectrin-likerepeats, and wherein n is an even number that is less than 24 and atleast 4. In some embodiments, the present invention provides nucleicacid encoding a mini-dystrophin peptide, wherein the mini-dystrophinpeptide comprises n spectrin-like repeats, wherein the mini-dystrophinpeptide contains no more than n spectrin-like repeats, and wherein n isan even number that is less than 24 and at least 4.

In some embodiments, n is 20 or less. In other embodiments, n is 16 orless. In particular embodiments, n is 12 or less. In additionalembodiments, n is 8 or less. In preferred embodiments, n is 4. Incertain embodiments, n is selected from 4, 8, 10, 12, 14, 16, 18, 20 and22. In some embodiments, the present invention provides compositionscomprising nucleic acid encoding a mini-dystrophin peptide, wherein themini-dystrophin peptide comprises a spectrin-like repeat domain, andwherein the spectrin-like repeat domain consists of n spectrin-likerepeats, wherein n is 4, 8, 12, 16, or 20. In certain embodiments, thepresent invention provides the peptides encoded by the nucleic acidsequences encoding the mini-dystrophin peptides.

In certain embodiments, the present invention provides compositionscomprising nucleic acid encoding a mini-dystrophin peptide, wherein themini-dystrophin peptide comprises i) a spectrin-like repeat domaincomprising 4 dystrophin spectrin-like repeats, ii) an actin-bindingdomain, and iii) a β-dystroglycan binding domain; and wherein themini-dystrophin peptide contains no more than 4 dystrophin spectrin-likerepeats.

In some embodiments, the present invention provides compositionscomprising a mini-dystrophin peptide, wherein the mini-dystrophinpeptide comprises a spectrin-like repeat domain comprising nspectrin-like repeats, wherein the mini-dystrophin peptide contains nomore than n spectrin-like repeats, and wherein n is an even number thatis less than 24 and at least 4. In particular embodiments, the presentinvention provides a cell (or cell line) containing the nucleic acid andpeptide sequences of the present invention.

In certain embodiments, the mini-dystrophin peptide is capable ofaltering a measurable muscle value in a DMD animal model by at leastapproximately 10% of the wild type value. In other embodiments, themini-dystrophin peptide is capable of altering a measurable muscle valuein a DMD animal model by at least approximately 20% of the wild typevalue. In particular embodiments, the mini-dystrophin peptide is capableof altering a measurable muscle value in a DMD animal model by at leastapproximately 30% of the wild type value. In preferred embodiments, themini-dystrophin peptide is capable of altering a measurable muscle valuein a DMD animal model to a level similar to the wild-type value (e.g.±4%). In certain embodiments, the nucleic acid comprises at least 2, orat least 4, spectrin-like repeat encoding sequences. In someembodiments, the spectrin-like repeat encoding sequences are precisespectrin-like repeat encoding sequences. In certain embodiments, thenucleic acid is less than 5 kilo-bases in length. In other embodiments,the nucleic acid is less than 6 kilo-bases in length. In particularembodiments, the nucleic acid comprises viral DNA (e.g. adenovirus DNA).In preferred embodiments, the viral DNA comprises adeno-associated viralDNA.

In certain embodiments, the present invention provides compositionscomprising nucleic acid encoding a mini-dystrophin peptide, wherein themini-dystrophin peptide comprises a spectrin-like repeat domain, andwherein the spectrin-like repeat domain consists of n spectrin-likerepeats, wherein n is an even number less than 24; and wherein thenucleic acid comprises an actin-binding domain encoding sequence, aβ-dystroglycan-binding domain encoding sequence, and at least 2, or atleast 4, spectrin-like repeat encoding sequences. In some embodiments,the nucleic acid comprises at least 4 spectrin-like repeat encodingsequences.

In certain embodiments, the present invention provides compositionscomprising nucleic acid, wherein the nucleic acid comprises at least 2spectrin-like repeat encoding sequences, and wherein the nucleic acidencodes a mini-dystrophin peptide comprising a spectrin-like repeatdomain, wherein the spectrin-like repeat domain consists of nspectrin-like repeats, and wherein n is an even number less than 24. Insome embodiments, the nucleic acid comprises at least 4 spectrin-likerepeat encoding sequences.

In some embodiments, the nucleic acid comprises SEQ ID NO:39 (i.e.ΔR4-R23). In other embodiments, the nucleic acid comprises SEQ ID NO:40(i.e. ΔR2-R21). In certain embodiments, the nucleic acid comprises SEQID NO:41 (i.e. ΔR2-R21+H3). In still other embodiments, the nucleic acidcomprises SEQ ID NO:42 (i.e. ΔH2-R19).

In certain embodiments, the nucleic acid comprises an expression vector(e.g. plasmid, virus, etc). In some embodiments, the expression vectorcomprises viral DNA. In certain embodiments, the viral DNA comprisesadeno-viral DNA. In some embodiments, the viral DNA comprises lentiviralDNA. In other embodiments, the viral DNA comprises helper-dependentadeno-viral DNA. In preferred embodiments, the viral DNA comprisesadeno-associated viral DNA. In some embodiments, the nucleic acid isinserted in a virus (e.g. adeno-associated virus, adenovirus,helper-dependent adeno-associated virus, lentivirus).

In certain embodiments, the nucleic acid comprises an actin-bindingdomain encoding sequence. In particular embodiments, the actin bindingdomain comprises at least a portion of SEQ ID NO:6 (e.g. 5%, 10%, 20%,40%, 50%, or 75% of SEQ ID NO:6). In other embodiments, the actinbinding domain comprises at least a portion of a homolog or mutatedversion of SEQ ID NO:6 (e.g. 5%, 10%, 20%, 40%, 50%, or 75% of a SEQ IDNO:6 homolog or mutated version of SEQ ID NO:6). In certain embodiments,the nucleic acid comprises a β-dystroglycan binding domain. In certainembodiments, the β-dystroglycan binding domain comprises at least aportion of a dystrophin hinge 4 encoding sequence (e.g. the 3′ 50% ofSEQ ID NO:34), and at least a portion of dystrophin cysteine-rich domainencoding sequence (e.g. the 5′ 75% of SEQ ID NO:35). In particularembodiments, at least a portion of hinge 4 is the WW domain (SEQ IDNO:45), or a homolog or mutation thereof.

In particular embodiments, the spectrin-like repeat encoding sequencesare selected from the group consisting of SEQ ID NOS:8-10, 12-27, and29-33. In some embodiments, the spectrin-like repeat encoding sequencesare selected from the group consisting of SEQ ID NOS:8-10, 12-27, and29-33, and homologs or mutations of SEQ ID NOS:8-10, 12-27, and 29-33.In preferred embodiments, the spectrin-like repeat encoding sequencesare selected from the group consisting of SEQ ID NOS:8-10 and 29-33. Insome embodiments, the spectrin-like repeat encoding sequences areidentical (e.g. all the sequences are SEQ ID NO:8). In preferredembodiments, the spectrin-like repeat encoding sequences are alldifferent (e.g. the nucleic acid sequence has only 4 spectrin-likerepeat encoding sequences, and these 4 are: SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:33). In certain embodiments, nucleic acidsequence comprises at least one spectrin-like repeat encoding sequenceselected from the group consisting of SEQ ID NOS:8-10, and at least onespectrin-like repeat encoding sequence selected from the groupconsisting of SEQ ID NOS:29-33.

In certain embodiments, the nucleic acid (or the resulting peptide)comprises at least one dystrophin hinge region. In some embodiments, thenucleic acid comprises at least one dystrophin hinge region selectedfrom hinge region 1, hinge region 2, hinge region 3 and hinge region 4.In some embodiments, the nucleic acid comprises at least one dystrophinhinge region selected from hinge region 1, hinge region 2, and hingeregion 3. In particular embodiments, dystrophin hinge region 1 is SEQ IDNO:7, or a homolog (See, e.g. FIG. 11), or a mutant version thereof. Inparticular embodiments, dystrophin hinge region 2 is SEQ ID NO:11, or ahomolog (See, e.g. FIG. 11), or a mutant version thereof. In certainembodiments, dystrophin hinge region 3 is SEQ ID NO:28, or a homolog(See, e.g. FIG. 11), or a mutant version thereof. In other embodiments,dystrophin hinge region 4 is SEQ ID NO:34, or a homolog (See, e.g. FIG.11), or a mutant version thereof.

In some embodiments, the nucleic acid comprises a sequence encoding atleast a portion of wild-type dystrophin C-terminal protein. In otherembodiments, the nucleic acid comprises at least a portion of the 5′untranslated region. In particular embodiments, the nucleic acidcomprises at least a portion of the 3′ untranslated region. In differentembodiments, the nucleic acid sequence comprises regulatory sequences(e.g. MCK enhancer and promoter elements). In particular embodiments,the nucleic acid sequence is operably linked to regulatory sequences(e.g. MCK enhancer and promoter elements). In certain embodiments, thenucleic acid sequence comprises a mutant muscle-specific enhancerregion.

In particular embodiments, the nucleic acid has less than 75% of a wildtype dystrophin 5′ untranslated region. In other embodiments, thenucleic acid has less than 50% or 20% or 1% (e.g. 0, 1, 2 nucleotidesfrom a wild type dystrophin 5′ untranslated region). In particularlypreferred embodiments, the nucleic acid sequence does not contain any ofthe wild-type dystrophin 5′ untranslated region. In certain embodiments,the nucleic acid has less than 75% of a wild type dystrophin 3′untranslated region. In other embodiments, the nucleic acid has lessthan 50%, preferably less than 40%, more preferably less than 35% of awild type dystrophin 3′ untranslated region. In certain embodiments, thenucleic acid does not contain a wild-type dystrophin 3′ untranslatedregion (or, in some embodiments, any type of 3′ untranslated region).

In particular embodiments, the mini-dystrophin peptide (e.g. encoded bythe nucleic acid of the present invention) comprises a substantiallydeleted dystrophin C-terminal domain. In some embodiments, themini-dystrophin peptide comprises less than 40% of wild type dystrophinC-terminal domain, preferably less than 30%, more preferably less than20%, even more preferably less than 1%, and most preferablyapproximately 0% (e.g. 0, 1, 2, 3 or 4 amino acids from the wild typedystrophin C-terminal domain). In some embodiments, the nucleic acidsequence comprises at least one intron sequence.

In some embodiments, the present invention provides methods forexpressing a mini-dystrophin peptide in a target cell, comprising; a)providing; i) a vector comprising nucleic acid encoding amini-dystrophin peptide, wherein the mini-dystrophin peptide comprises aspectrin-like repeat domain, and wherein the spectrin-like repeat domainconsists of n spectrin-like repeats, wherein n is an even number lessthan 24, and ii) a target cell, and b) contacting the vector with thetarget cell under conditions such that the mini-dystrophin peptide isexpressed in the target cells. In certain embodiments, the contactingcomprises transfecting. In some embodiments, the contacting is performedin-vitro. In particular embodiments, the contacting is done in-vivo. Inother embodiments, the target cell is a muscle cell. In particularembodiments, the target cell further comprises a subject (e.g. withDuchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD)).In preferred embodiment, the mini-dystrophin peptide is expressed in thecells of a subject (e.g. such that symptoms of DMD or BMD are reduced oreliminated).

In certain embodiments, the present invention provides methodscomprising; a) providing; i) a vector comprising nucleic acid encoding amini-dystrophin peptide, wherein the mini-dystrophin peptide comprises aspectrin-like repeat domain comprising n spectrin-like repeats, whereinthe mini-dystrophin peptide contains no more than n spectrin-likerepeats, and wherein n is an even number that is less than 24 and atleast 4, and ii) a subject comprising a target cells (e.g. a subjectwith symptoms of a muscle disease, such as Muscular Dystrophy); and b)contacting the vector with the subject under conditions such that themini-dystrophin peptide is expressed in the target cell (e.g. such thatthe symptoms are reduced or eliminated). In preferred embodiments, thenucleic acid encoding the mini-dystrophin peptide is contained within anviral vector (e.g. adeno-associated viral vector), and the contacting isdone by means of injecting the viral vector into the subject.

In particular embodiments, the present invention provides compositionscomprising nucleic acid, wherein the nucleic acid encodes amini-dystrophin peptide, and wherein the mini-dystrophin peptidecomprises a substantially deleted dystrophin C-terminal domain. In someembodiments, the present invention provides the peptides encoded by thenucleic acid of the present invention. In certain embodiments, thesubstantially deleted dystrophin C-terminal domain is less than 40% of awild type dystrophin C-terminal domain. In other embodiments, thesubstantially deleted dystrophin C-terminal domain is less than 30%,20%, or 1% of a wild type dystrophin C-terminal domain. In preferredembodiments, the substantially deleted dystrophin C-terminal domain isapproximately 0% of a wild type dystrophin C-terminal domain. In certainembodiments, the mini-dystrophin peptide does not contain any portion ofthe wild type dystrophin C-terminal domain (i.e. it is completelydeleted).

In certain embodiments, the mini-dystrophin peptide is capable ofaltering a measurable muscle value in a DMD animal model by at least 10%of the wild type value. In other embodiments, the mini-dystrophinpeptide is capable of altering a measurable muscle value in a DMD animalmodel by at least 20% of the wild type value. In particular embodiments,the mini-dystrophin-peptide is capable of altering a measurable musclevalue in a DMD animal model by at least 30% of the wild type value. Inpreferred embodiments, the mini-dystrophin peptide is capable ofaltering a measurable muscle value in a DMD animal model to a levelsimilar to the wild-type value (e.g. ±4%).

In certain embodiments, the nucleic acid comprises an expression vector(e.g. plasmid, virus, etc). In some embodiments, the expression vectorcomprises viral DNA. In certain embodiments, the viral DNA comprisesadeno-viral DNA. In some embodiments, the viral DNA comprises lentiviralDNA. In other embodiments, the viral DNA comprises helper-dependentadeno-viral DNA. In preferred embodiments, the viral DNA comprisesadeno-associated viral DNA. In some embodiments, the nucleic acid isinserted in a virus (e.g. adeno-associated virus, adenovirus,helper-dependent adeno-associated virus, lentivirus).

In certain embodiments, the nucleic acid comprises an actin-bindingdomain encoding sequence. In particular embodiments, the actin bindingdomain comprises at least a portion of SEQ ID NO:6 (e.g. 5%, 10%, 20%,40%, 50%, or 75% of SEQ ID NO:6). In other embodiments, the actinbinding domain comprises at least a portion of a homolog or mutatedversion of SEQ ID NO:6 (e.g. 5%, 10%, 20%, 40%, 50%, or 75% of a SEQ IDNO:6 homolog or mutated version of SEQ ID NO:6). In certain embodiments,the nucleic acid comprises a β-dystroglycan binding domain. In certainembodiments, the β-dystroglycan binding domain comprises at least aportion of a dystrophin hinge 4 encoding sequence (e.g. the 3′ 50% ofSEQ ID NO:34), and at least a portion of dystrophin cysteine-rich domainencoding sequence (e.g. the 5′ 75% of SEQ ID NO:35). In particularembodiments, at least a portion of hinge 4 is the WW domain (SEQ IDNO:45), or a homolog or mutation thereof.

In certain embodiments, the nucleic acid comprises at least onedystrophin hinge region. In some embodiments, the nucleic acid comprisesat least one dystrophin hinge region selected from hinge region 1, hingeregion 2, hinge region 3 and hinge region 4. In some embodiments, thenucleic acid comprises at least one dystrophin hinge region selectedfrom hinge region 1, hinge region 2, and hinge region 3. In particularembodiments, dystrophin hinge region 1 is SEQ ID NO:7, or a homolog(See, e.g. FIG. 11), or a mutant version thereof. In particularembodiments, dystrophin hinge region 2 is SEQ ID NO:11, or a homolog(See, e.g. FIG. 11), or a mutant version thereof. In certainembodiments, dystrophin hinge region 3 is SEQ ID NO:28, or a homolog(See, e.g. FIG. 11), or a mutant version thereof. In other embodiments,dystrophin hinge region 4 is SEQ ID NO:34, or a homolog (See, e.g. FIG.11), or a mutant version thereof.

In other embodiments, the nucleic acid comprises at least a portion ofthe 5′ untranslated region. In particular embodiments, the nucleic acidcomprises at least a portion of the 3′ untranslated region. In differentembodiment, the nucleic acid sequence comprises regulatory sequences(e.g. MCK enhancer and promoter elements). In particular embodiments,the nucleic acid sequence is operably linked to regulatory sequences(e.g. MCK enhancer and promoter elements). In certain embodiments, thenucleic acid sequence comprises a mutant muscle-specific enhancerregion.

In particular embodiments, the nucleic acid contains less that 75% of awild type dystrophin 5′ untranslated region. In other embodiments, thenucleic acid contains less than 50% or 20% or 1% (e.g. 0, 1, 2nucleotides from a wild type dystrophin 5′ untranslated region). Inparticularly preferred embodiments, the nucleic acid sequence does notcontain any of the wild-type dystrophin 5′ untranslated region. Incertain embodiments, the nucleic acid has less than 75% of a wild typedystrophin 3′ untranslated region. In other embodiments, the nucleicacid has less than 50%, preferably less than 40%, more preferably lessthan 35% of a wild type dystrophin 3′ untranslated region. In certainembodiments, the nucleic acid does not contain a wild-type dystrophin 3′untranslated region (or, in some embodiments, any type of 3′untranslated region).

In some embodiments, the present invention provides methods forexpressing a mini-dystrophin peptide in a target cell, comprising; a)providing; i) a vector comprising nucleic acid, wherein the nucleic acidencodes a mini-dystrophin peptide comprising a substantially deleteddystrophin C-terminal domain, and ii) a target cell, and b) contactingthe vector with the target cell under conditions such that themini-dystrophin peptide is expressed in the target cells. In certainembodiments, the contacting comprises transfecting. In otherembodiments, the target cell is a muscle cell.

In certain embodiments, the present invention provides systems and kitswith the mini-dystrophin nucleic acid and/or peptide sequences describedherein. In certain embodiments, the systems and kits of the presentinvention comprise a nucleic acid sequence encoding a mini-dystrophinpeptide (and/or the mini-dystrophin peptide) and one other component(e.g. an insert component with written instructions for using themini-dystrophin nucleic acid, or a nucleic acid encoding a vector, or acomponent for delivering the nucleic acid to a subject, cells forexpressing the mini-dystrophin peptide, a buffer, etc.). In certainembodiments, the present invention provides a computer readable medium(e.g. CD, hard drive, floppy disk, magnetic tape, etc.) that containsthe nucleic acid or amino acid sequences of the present invention (e.g.a computer readable representation of the nucleotide bases used to makea mini-dystrophin nucleic acid sequence).

In some embodiments, the present invention provides mini-dystrophinnucleic acid sequences for use as a medicament. In other embodiments,the present invention provides mini-dystrophin peptides for use as amedicament. In particular embodiments, the present invention providesthe use of mini-dystrophin nucleic acid sequences for preparing a drugfor a therapeutic application. In additional embodiments, the presentinvention provides the use of mini-dystrophin peptides for preparing adrug for a therapeutic application. In some embodiments, the presentinvention provides mini-dystrophin nucleic acid sequences for thepreparation of a composition for the treatment of a muscle disease (e.g.DMD). In other embodiments, the present invention providesmini-dystrophin peptides for the preparation of a composition for thetreatment of a muscle disease (e.g. DMD).

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence for wild-type human dystrophincDNA.

FIG. 2 shows the nucleic acid sequence for wild-type mouse dystrophincDNA.

FIG. 3 shows the nucleic acid sequence for wild-type human utrophincDNA.

FIG. 4 shows the nucleic acid sequence for wild-type mouse utrophin cDNA

FIG. 5 shows various domains of the nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 6 shows various domains of the nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 7 shows various domains of the nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 8 shows various domains of the nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 9 shows various domains of the nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 10 shows the 3′ UTR domain nucleic acid sequence for wild-typehuman dystrophin cDNA.

FIG. 11 shows a sequence alignment between wild-type human dystrophincDNA (bases 1220-9328 of SEQ ID NO:1) and wild-type mouse dystrophincDNA (bases 1238-9319 of SEQ ID NO:2). The various domains in the humandystrophin sequence have spaces betwcen them with the ends highlightedin bold. In this regard, homologous sequences for various domains in themouse cDNA sequence are seen.

FIG. 12 shows the nucleic acid sequence for ΔR4-R23, a nucleic acidsequence encoding a mini-dystrophin peptide.

FIG. 13 shows the nucleic acid sequence for ΔR2-R21, a nucleic acidsequence encoding a mini-dystrophin peptide.

FIG. 14 shows the nucleic acid sequence for ΔR2-R21+H3, a nucleic acidsequence encoding a mini-dystrophin peptide.

FIG. 15 shows the nucleic acid sequence for ΔH2-R19, a nucleic acidsequence encoding a mini-dystrophin peptide.

FIG. 16 shows the complete cDNA sequence for human skeletal muscle alphaactinin.

FIG. 17 shows the nucleic acid sequence for ΔR9-R16, a nucleic acidsequence encoding a mini-dystrophin peptide.

FIG. 18 shows the nucleic acid sequence for the WW domain.

FIG. 19 shows various transgenic expression constructs tested in Example1.

FIG. 20 shows the contractile properties of EDL, soleus, and diaphragmmuscles in wild-type, mdx, and dystrophin Δ71-78 mice.

FIG. 21 show the nucleic acid sequence for pBSX.

FIG. 22 shows a restriction map for pBSX.

FIG. 23 shows the ‘full-length’ HDMD sequence.

FIG. 24 shows the cloning procedure for ΔR4-R23.

FIG. 25 shows the cloning procedure for ΔR2-R21+H3.

FIG. 26 shows the cloning procedure for ΔR2-R21.

FIG. 27 shows a schematic illustration of the domains encoded by thetruncated and full-length dystrophin sequences tested in Example 5.

FIG. 28 is a graph showing the percentage of myofibers in quadricepmuscles of 3 month old mice that display centrally-located nuclei in theindicated strains of transgenic mice.

FIG. 29 shows graphs depicting the force generating capacity indiaphragm (A) or EDL (B) muscles of the indicated strains of dystrophintransgenic mdx mice and control mice.

FIG. 30 shows a graph depicting the force generating capacity in EDL (A)or diaphragm (B) muscles of the indicated strains of dystrophintransgenic mdx mice and control mice.

FIG. 31 is a graph showing the percentage of force generating capacitylost after 1 or 2 lengthening contractions of the tibialis anteriormuscle of the indicated strains of dystrophin transgenic mdx mice andcontrol mice.

FIG. 32 is a graph showing the total distance run on a treadmill byanimals from the indicated strains of dystrophin transgenic mdx mice andcontrol mice.

FIG. 33 shows a graph depicting the total body mass (A) and mass of thetibialis anterior muscle (B) of the indicated strains of dystrophintransgenic mdx mice and control mice.

FIG. 34 is a schematic illustration of the structure of amini-dystrophin expression cassette inserted into an adeno-associatedviral vector.

FIG. 35 is a schematic illustration of the structure of plasmid pTZ19R(top) and the sequence of the multiple cloning site in the vector(bottom) (SEQ ID NOS:95 and 96).

FIG. 36 shows the nucleic acid sequence of various MCK enhancer regions(wild-type and mutant).

FIG. 37 shows the nucleic acid sequence of various MCK promoter regions.

FIG. 38 shows a comparison between domains in dystrophin and utrophin.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “measurable muscle values” refers tomeasurements of dystrophic symptoms (e.g. fibrosis, an increasedproportion of centrally located nuclei, reduced force generation byskeletal muscle, etc.) in an animal. These measurements may be taken,for example, to determine the wild-type value (i.e. the value in acontrol animal), to determine the value in a DMD (Duchenne musculardystrophy) animal model (e.g. in an mdx mouse model), and to determinethe value in a DMD animal model expressing the mini-dystrophin peptidesof the present invention. Various assays may be employed to determinemeasurable muscle values in an animal including, but not limited to,assays measuring fibrosis, phagocytic infiltration of muscle tissue,variation in myofiber size, an increased proportion of myofibers withcentrally located nuclei, elevated serum levels of muscle pyruvatekinase, contractile properties assays, DAP (dystrophin associatedprotein) assays, susceptibility to contraction induced injuries andmeasured force assays (See Examples 1 and 4).

As used herein, the term “mini-dystrophin peptide” refers to a peptidethat is smaller in size than the full-length wild-type dystrophinpeptide, and that is capable of altering (increasing or decreasing) ameasurable muscle value in a DMD animal model by at least approximately10% such that the value is closer to the wild-type value (e.g. a mdxmouse has a measurable muscle value that is 50% of the wild-type value,and this value is increased to at least 60% of the wild-type value; or amdx mouse has a measurable muscle value that is 150% of the wild-typevalue, and this value is decreased to at most 140% of the wild-typevalue). In some embodiments, the mini-dystrophin-peptide is capable ofaltering a measurable muscle value in a DMD animal model by at leastapproximately 20% of the wild type value. In certain embodiments, themini-dystrophin-peptide is capable of altering a measurable muscle valuein a DMD animal model by at least approximately 30% of the wild typevalue. In preferred embodiments, the mini-dystrophin peptide is capableof altering a measurable muscle value in a DMD animal model to a levelsimilar to the wild-type value (e.g. ±4%).

As used herein, the term “wild-type dystrophin cysteine-rich domain”refers to a peptide encoded by the nucleic acid sequences in SEQ IDNO:35 (e.g. in human), as well as wild type peptide homologs encoded bynucleic acid homologs of SEQ ID NO:35 (See, FIG. 11).

As used herein, the term “wild type dystrophin C-terminal domain” refersto a peptide encoded by the nucleic acid sequences in SEQ ID NO:36 (e.g.in human), as well as wild type peptide homologs encoded by nucleic acidhomologs of SEQ ID NO:36 (See, FIG. 11).

As used herein, the term “mini-dystrophin peptide comprising asubstantially deleted dystrophin C-terminal domain” refers to amini-dystrophin peptide that has less than 45% of a wild type dystrophinC-terminal domain. In some embodiments, the mini-dystrophin peptidecomprises less than 40% of wild type dystrophin C-terminal domain,preferably less than 30%, more preferably less than 20%, even morepreferably less than 1%, and most preferably approximately 0% (e.g. 0,1, 2, 3 or 4 amino acids from the wild type dystrophin C-terminaldomain). The construction of mini-dystrophin peptides with asubstantially deleted dystrophin C-terminal domain may be accomplished,for example, by deleting all or a portion of SEQ ID NO:36 from humandystrophin SEQ ID NO:1 (See, e.g. Example 3C).

As used herein, the term “wild type dystrophin 5′ untranslated region”refers to the nucleic acid sequence at the very 5′ end of a wild typedystrophin nucleic acid sequence (e.g. SEQ ID NOS:1 and 2) thatimmediately precedes the amino acid coding regions. For example, forhuman dystrophin, SEQ ID NO:5 (the first 208 bases) is the 5′untranslated region (a homolog in mouse may be seen in FIG. 11).

As used herein, the term “wild type dystrophin 3′ untranslated region”refers to the nucleic acid sequence at the very 3′ end of a wild typedystrophin nucleic acid sequence (e.g. SEQ ID NOS:1 and 2) thatimmediately proceeds the amino acid coding regions. For example, forhuman dystrophin, SEQ ID NO:38 (the last 2690 bases of the humandystrophin gene) is the 3′ untranslated region (a homolog in mouse maybe seen in FIG. 11).

As used herein, the term “actin-binding domain encoding sequence” refersto the portion of a dystrophin nucleic sequence that encodes apeptide-domain capable of binding actin in vitro (e.g. SEQ ID NO:6), aswell as homologs (See, FIG. 11), conservative mutations, and truncationsof such sequences that encode peptide-domains that are capable ofbinding actin in vivo. Determining whether a particular nucleic acidsequence encodes a peptide-domain (e.g. homolog, mutation, or truncationof SEQ ID NO:6) that will bind actin in vitro may be performed, forexample, by screening the ability of the peptide-domain to bind actin invitro in a simple actin binding assay (See, Corrado et al., FEBSLetters, 344:255-260 [1994], describing the expression of candidatedystrophin peptides as fusion proteins, absorbing F-actin on tomicrotiter plates, incubating the candidate peptides in the F-actincoated microtiter plates, washing the plates, adding anti-fusion proteinrabbit antibody, and adding an anti-rabbit antibody conjugated to adetectable marker).

As used herein, the term “β-dystroglycan-binding domain encodingsequence” refers to the portion of a dystrophin nucleic sequence thatencodes a peptide-domain capable of binding β-dystroglycan in vivo (e.g.SEQ ID NOS:34 and 35), as well as homologs (See, FIG. 11), conservativemutations, and truncations of such sequences that encode peptide-domainsthat are capable of binding β-dystroglycan in vivo. In preferredembodiments, the β-dystroglycan-binding domain encoding sequenceincludes at least a portion of a hinge 4 encoding region (e.g. SEQ IDNO:45, the WW domain) and at least a portion of a wild-type dystrophincysteine-rich domain (e.g. at least a portion of SEQ ID NO:35) (See,e.g. Jung et al., JBC, 270 (45):27305 [1995]). Determining whether aparticular nucleic acid sequence encodes a peptide-domain (e.g. homolog,mutation, or truncation) that will bind β-dystroglycan in vivo may beperformed, for example, by first screening the ability of thepeptide-domain to bind β-dystroglycan in vitro in a simpleβ-dystroglycan binding assay (See, Jung et al., pg 27306—constructingpeptide-domain dystrophin-GST fusion peptides and radioactively labelledβ-dystroglyean, immobilizing the fusion proteins on glutathione-agarosebeads, incubating the beads with the radioactively labelledβ-dystroglycan, pelleting the beads, washing the beads, and resolvingthe sample on an SDS-polyacrylamide gel, staining with Coomasie blue,exposing to film, and quantifying the amount of radioactivity present).Nucleic acid sequences found to express peptides capable of bindingβ-dystroglycan in such assays may then, for example, be tested in vivoby transfecting a cell line (e.g., COS cells) with two expressionvectors, one expressing the dystroglycan peptide and the otherexpressing the candidate peptide domain (as a fusion protein). Afterculturing the cells, the protein is then extracted and aco-immunoprecipitation is performed for one of the proteins, followed bya Western blot for the other.

As used herein, the term “spectrin-like repeats” refers to peptidescomposed of approximately 100 amino acids that are responsible for therod-like shape of many structural proteins including, but not limitedto, dystrophin, utrophin, fodrin, alpha-actin, and spectrin, when thespectrin-like repeats are present in multiple copies (e.g.dystrophin-24, utrophin-22, alpha-actin-4, spectrin-16, etc).Spectrin-like repeats also refers to mutations of these naturalpeptides, such as conservative changes in amino acid sequence, as wellas the addition or deletion of up to 5 amino acids to/from the end of aspectrin-like repeat. Spectrin-like repeats includes ‘precisespectrin-like repeats’ (see below). Examples of spectrin-like repeatsinclude, but are not limited to, peptides encoded by nucleic acidsequences found in wild-type human dystrophin (e.g. SEQ ID NOS:8-10,12-27, and 29-33).

As used herein, the term “spectrin-like repeat encoding sequences”refers to nucleic acid sequences encoding spectrin-like repeat peptides.This term includes natural and synthetic nucleic acid sequences encodingthe spectrin-like repeats (e.g. both the naturally occurring and mutatedspectrin-like repeat peptides). Examples of spectrin-like repeatencoding sequences include, but are not limited to, SEQ ID NOS:8-10,12-27, and 29-33.

As used herein, the term “precise spectrin-like repeat encodingsequences” refers to nucleic acid sequences encoding spectrin-likerepeat peptides with up to 1 additional amino acid added to, or deletedfrom, the spectrin-like repeat.

As used herein, the term “spectrin-like repeat domain” refers to theregion in a mini-dystrophin peptide that contains the spectrin-likerepeats of the mini-dystrophin peptide.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired enzymatic activity is retained. The term “gene” encompasses bothcDNA and genomic forms of a given gene.

The tern “wild-type” refers to a gene, gene product, or other sequencethat has the characteristics of that gene or gene product when isolatedfrom a naturally occurring source. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” refers to a gene, gene product, or othersequence that displays modifications in sequence and or functionalproperties (e.g. altered characteristics) when compared to the wild-typegene or gene product. It is noted that naturally-occurring mutants canbe isolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotide, usuallymore than three (3), and typically more than ten (10) and up to onehundred (100) or more (although preferably between twenty and thirty).The exact size will depend on many factors, which in turn depends on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, or a combination thereof.

As used herein, the term “regulatory sequence” refers to a geneticsequence or element that controls some aspect of the expression ofnucleic acid sequences. For example, a promoter is a regulatory elementthat facilitates the initiation of transcription of an operably linkedcoding region. Other regulatory elements are enhancers, splicingsignals, polyadenylation signals, termination signals, etc. Examplesinclude, but are not limited to, the 5′ UTR of the dystrophin gene (SEQID NO:5), MCK promoters and enhancers (both wild type and mutant, SeeU.S. provisional app. Ser No. 60/218,436, filed Jul. 14, 2000, andInternational Application PCT/US01/22092, filed Jul. 13, 2001, both ofwhich are hereby incorporated by reference).

Transcriptional control signals in eucaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription. The present invention contemplates modified enhancerregions.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired coding sequence and appropriate DNA sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism (e.g., mammal). DNA sequences necessary forexpression in procaryotes include a promoter, optionally an operatorsequence, a ribosome binding site and possibly other sequences.Eukaryotic cells are known to utilize promoters, polyadenlyation signalsand enhancers.

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

“Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be detected). Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon.

The “complement” of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Complementarity need not be perfect;stable duplexes may contain mismatched base pairs or unmatched bases.Those skilled in the art of nucleic acid technology can determine duplexstability empirically considering a number of variables including, forexample, the length of the oligonucleotide, base composition andsequence of the oligonucleotide, ionic strength and incidence ofmismatched base pairs.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (ie., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target thatlacks even a partial degree of complementarity (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCL, 6.9 g/lNaH₂PO₄—H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDA,5× Denhardt's reagent [50× Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V, Sigma)] and 100 μg/mldenatured salmon sperm DNA followed by washing in solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides inlength is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprises conditions equivalent to binding or hybridizingat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCL, 6.9 g/lNaH₂PO₄—H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA, followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides is employed.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “muscle cell” refers to a cell derived frommuscle tissue, including, but not limited to, cells derived fromskeletal muscle, smooth muscle (e.g. from the digestive tract, urinarybladder, and blood vessels), and cardiac muscle. The term includesmuscle cells in vitro, ex vivo, and in vivo. Thus, for example, anisolated cardiomyocyte would constitute a muscle cell, as would a cellas it exists in muscle tissue present in a subject in vivo. This termalso encompasses both terminally differentiated and nondifferentiatedmuscle cells, such as myocytes, myotubes, myoblasts, cardiomyocytes, andcardiomyoblasts.

As used herein, the term “muscle-specific” in reference to an regulatoryelement (e.g. enhancer region, promoter region) means that thetranscriptional activity driven by these regions is mostly in musclecells or tissue (e.g. 20:1) compared to the activity conferred by theregulatory sequences in other tissues. An assay to determine themuscle-specificity of a regulatory region is provided in Example 5 below(measuring beta-galactoside in muscle cells and liver cells from a mousetransfected with an expression vector).

As used herein, the term “mutant muscle-specific enhancer region” refersto a wild-type muscle-specific enhancer region that has been modified(e.g. deletion, insertion, addition, substitution), and in particular,has been modified to contain an additional MCK-R control element (SeeU.S. Prov. App. Ser. No. 60/218,436, hereby incorporated by reference,and section IV below).

DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for expressingmini-dystrophin peptides. In particular, the present invention providescompositions comprising nucleic acid sequences that are shorter thanwild-type dystrophin cDNA and that express mini-dystrophin peptides thatfunction in a similar manner as wild-type dystrophin proteins. Thepresent invention also provides compositions comprising mini-dystrophinpeptides, and methods for expressing mini-dystrophin peptides in targetcells.

The present invention provides such shortened nucleic acid sequences(and resulting peptides) in a variety of ways. For example, the presentinvention provides nucleic acid encoding only 4, 8, 12, 16, and 20spectrin-like repeat encoding sequences (i.e. nucleic acid encoding anexact number of spectrin-like repeats that are multiples of 4). Aswild-type dystrophin has 24 spectrin-like repeat encoding sequences,providing nucleic acid encoding fewer numbers of repeats reduces thesize of the dystrophin gene (e.g. allowing the nucleic acid sequence tofit into vectors with limited cloning capacity). Another example of suchshortened nucleic acid sequences are those that lack at least a portionof the carboxy-terminal domain of wild-type dystrophin nucleic acid. Afurther example of such shortened nucleic acid sequences are those thatlack at least a portion of the 3′ untranslated region, or 5′untranslated region, or both.

I. Dystrophin

A. Dystrophin Structure

In some embodiments, the present invention provides gene constructscomprising spectrin-like repeats from human dystrophin. Dystrophin is a427 kDa cytoskeletal protein and is a member of the spectrin/αactininsuperfamily (See e.g., Blake et al., Brain Pathology, 6:37 [1996];Winder, J. Muscle Res. Cell. Motil., 18:617 [1997]; and Tinsley el al.,PNAS, 91:8307 [1994]). The N-terminus of dystrophin binds to actin,having a higher affinity for non-muscle actin than for sarcomeric actin.Dystrophin is involved in the submembraneous network of non-muscle actinunderlying the plasma membrane. Dystrophin is associated with anoligomeric, membrane spanning complex of proteins and glycoproteins, thedystrophin-associated protein complex (DPC). The N-terminus ofdystrophin has been shown in vitro to contain a functional actin-bindingdomain. The C-terminus of dystrophin binds to the cytoplasmic tail ofβ-dystroglycan, and in concert with actin, anchors dystrophin to thesarcolemma. Also bound to the C-terminus of dystrophin are thecytoplasmic members of the DPC. Dystrophin thereby provides a linkbetween the actin-based cytoskeleton of the muscle fiber and theextracellular matrix. It is this link that is disrupted in musculardystrophy.

The central rod domain of dystrophin is composed of a series of 24weakly repeating units of approximately 110 amino acids, similar tothose found in spectrin (i.e., spectrin-like repeats). This domainconstitutes the majority of dystrophin and gives dystrophin a flexiblerod-like structure. The rod-domain is interrupted by four hinge regionsthat are rich in proline. It is contemplated that the rod-domainprovides a structural link between member of the DPC. Table 1 shows anoverview of the structural and functional domains of human dystrophin.

TABLE 1 Full Length Human Dystrophin cDNA Nucleotides Feature SEQ ID NO: 1-208 5′ untranslated region SEQ ID NO:5  209-211 Start codon (ATG) —209-964 N terminus SEQ ID NO:6   965-1219 Hinge 1 SEQ ID NO:7  1220-1546Spectrin-like repeat No. 1 SEQ ID NO:8  1547-1879 Spectrin-like repeatNo. 2 SEQ ID NO:9  1880-2212 Spectrin-like repeat No. 3 SEQ ID NO:102213-2359 Hinge 2 SEQ ID NO:11 2360-2692 Spectrin-like repeat No. 4 SEQID NO:12 2693-3019 Spectrin-like repeat No. 5 SEQ ID NO:13 3020-3346Spectrin-like repeat No. 6 SEQ ID NO:14 3347-3673 Spectrin-like repeatNo. 7 SEQ ID NO:15 3674-4000 Spectrin-like repeat No. 8 SEQ ID NO:164001-4312 Spectrin-like repeat No. 9 SEQ ID NO:17 4313-4588Spectrin-like repeat No. 10 SEQ ID NO:18 4589-4915 Spectrin-like repeatNo. 11 SEQ ID NO:19 4916-5239 Spectrin-like repeat No. 12 SEQ ID NO:205340-5551 Spectrin-like repeat No. 13 SEQ ID NO:21 5552-5833Spectrin-like repeat No. 14 SEQ ID NO:22 5834-6127 Spectrin-like repeatNo. 15 SEQ ID NO:23 6128-6187 20 amino acid insert (not hinge) —6188-6514 Spectrin-like repeat No. 16 SEQ ID NO:24 6515-6835Spectrin-like repeat No. 17 SEQ ID NO:25 6836-7186 Spectrin-like repeatNo. 18 SEQ ID NO:26 7187-7489 Spectrin-like repeat No. 19 SEQ ID NO:277490-7612 Hinge 3 SEQ ID NO:28 7613-7942 Spectrin-like repeat No. 20 SEQID NO:29 7943-8269 Spectrin-like repeat No. 21 SEQ ID NO:30 8270-8617Spectrin-like repeat No. 22 SEQ ID NO:31 8618-9004 Spectrin-like repeatNo. 23 SEQ ID NO:32 9005-9328 Spectrin-like repeat No. 24 SEQ ID NO:339329-9544 Hinge 4 SEQ ID NO:34  9545-10431 Start of C terminus SEQ IDNO:35 10432-11254 Alternatively spliced exons 71-78 SEQ ID NO:3611255-11266 End of Coding Region SEQ ID NO:37 11267-13957 3′untranslated region SEQ ID NO:38 *Domain structure based on Winder etal., Febs Letters, 369:27-33 (1995)

B. Spectrin-Like Repeats

Spectrin-like repeats are about 100 amino acids long and are found in anumber of proteins, including the actin binding proteins spectrin,fodrin, α-actinin, and dystrophin, but their function remains unclear(Dhermy, 1991. Biol. Cell, 71:249-254). These domains may be involved inconnecting functional domains and/or mediate protein-proteininteractions. The many tandem, spectrin-like motifs that comprise mostof the mass of the proteins in this superfamily are responsible fortheir similar flexible, rod-like molecular shapes. Although thesehomologous motifs are frequently called repeats or repetitive segments,adjacent segments in each protein are only distantly relatedevolutionarily.

Spectrin is a cytoskeletal protein of red blood cells that is associatedwith the cytoplasmic side of the lipid bilayer (See e.g., Speicher andUrsitti, Current Biology, 4:154 [1994]). Spectrin is a long-thinflexible rod-shaped protein that constitutes about 25% of themembrane-associated protein mass. Spectrin is composed of two largepolypeptide chains, α-spectrin (˜240 kDa) and β-spectrin (˜220 kDa) andserves to cross-link short actin oligomers to form a dynamictwo-dimensional submembrane latticework. Spectrin isoforms have beenfound in numerous cell types and have been implicated in a variety offunctions.

The recent determination of the crystal structure of a single domain ofspectrin provides insight into the structure function of an entire classof large actin cross-linking proteins (Yan et al., Science, 262:2027[1993]). The domain is an example of a spectrin-like repeat. Earlyanalysis of spectrin-like repeats by partial peptide sequence analysisdemonstrated that most of the antiparallel spectrin heterodimer is madeup of homologous 106 residue motifs. Subsequent sequence analyses ofcDNAs confirmed that this small motif is the major building block forall spectrin isoforms, as well as for the related actinins anddystrophins (Matsudaira, Trends Biochem Sci, 16:87 [1991]).

Given their similar sequences, all spectrin motifs are expected to haverelated, but not identical, three-dimensional structures. The structureof a single Drosophila spectrin motif, 14, which has now been determined(Yan et al., Science, 262:2027 [1993]), should therefore provide insightinto the overall conformation of spectrins in particular and, to a morelimited extent, the other members of the spectrin superfamily. Thestructure shows that the spectrin motif forms a three-helix bundle,similar to the earliest conformational prediction based on the analysisof multiple homologous motifs (Speicher and Marchesi, Nature, 311:177[1984]).

II. Variants and Homologs of Dystrophin

The present invention is not limited to the spectrin-like repeatencoding sequences SEQ ID NOS:8-10, 12-27, and 29-33, but specificallyincludes nucleic acid sequences capable of hybridizing to thespectrin-like repeat encoding sequences SEQ ID NOS:8-10, 12-27, and29-33, (e.g. capable of hybridizing under high stringent conditions).Those skilled in the art know that different hybridization stringenciesmay be desirable. For example, whereas higher stringencies may bepreferred to reduce or eliminate non-specific binding between thespectrin-like repeat encoding sequences SEQ ID NOS:8-10, 12-27, and29-33, and other nucleic acid sequences, lower stringencies may bepreferred to detect a larger number of nucleic acid sequences havingdifferent homologies to the nucleotide sequence of SEQ ID NOS:8-10,12-27, and 29-33.

Accordingly, in some embodiments, the dystrophin spectrin-like repeatsof the compositions of the present invention (e.g., SEQ ID NOs:8-10,12-27, and 29-33) are replaced with different spectrin-like repeats,including, but not limited to, variants, homologs, truncations, andadditions of dystrophin spectrin-like repeats. Candidate spectrin-likerepeats are screened for activity using any suitable assay, including,but not limited to, those described below and in illustrative Examples 1and 5.

A. Homologs

1. Dystrophin From other Species

In some embodiments, the spectrin-like repeats of the gene constructs ofthe present invention are replaced with spectrin-like repeats ofdystrophin from other species (e.g., homologs of dystrophin), including,but not limited to, those described herein. Homologs of dystrophin havebeen identified in a variety of organisms, including mouse (Genbankaccession number M68859); dog (Genbank accession number AF070485); andchicken (Genbank accession number X13369). The spectrin-like repeats ofthe mouse dystrophin gene were compared to the human gene (See FIG. 11)and were shown to have significant homology. Similar comparisons can begenerated with homologs from other species, including but not limitedto, those described above, by using a variety of available computerprograms (e.g., BLAST, from NCBI). Candidate homologs can be screenedfor biological activity using any suitable assay, including, but notlimited to, those described herein.

2. Utrophin

In some embodiments, the spectrin-like repeats of the gene constructs ofthe present invention are replaced with spectrin-like repeats fromanother peptide (e.g., homologs of dystrophin). For example, in someembodiments, spectrin-like repeats from the utrophin protein (See e.g.,Genbank accession number X69086; SEQ ID NO:3; FIG. 3) are utilized.Utrophin is an autosomally-encoded homolog of dystrophin and has beenpostulated that the proteins play a similar physiological role (For arecent review, See e.g., Blake et al., Brain Pathology, 6:37 [1996]).Human utrophin shows substantial homology to dystrophin, with the majordifference occurring in the rod domain, where utrophin lacks repeats 15and 19 and two hinge regions (See e.g., Love et al., Nature 339:55[1989]; Winder et al., FEBS Lett., 369:27 [1995]). Utrophin thuscontains 22 spectrin-like repeats and two hinge regions. A comparison ofthe rod domain of Utrophin and Dystrophin is shown in FIG. 38.

In addition, in some embodiments, spectrin-like repeats from a homologof utrophin are utilized. Homologs of utrophin have been identified in avariety of organisms, including mouse (Genbank accession number Y12229;SEQ ID NO:4; FIG. 4) and rat (Genbank accession number AJ002967). Thenucleic acid sequence of these or additional homologs can be compared tothe nucleic acid sequence of human utrophin using any suitable methods,including, but not limited to, those described above. Candidatespectrin-like repeats from human utrophin or utrophin homologs can bescreened for biological activity using any suitable assay, including,but not limited to, those described herein.

3. Alpha-actinin

In some embodiments, spectrin-like repeats from Dystrophin are replacedwith spectrin-like repeats from alpha-actinin. The microfilament proteinalpha-actinin exists as a dimer. The N-terminal regions of bothpolypeptides, arranged in antiparallel orientation, comprise theactin-binding regions, while the C-terminal, larger parts consist offour spectrin-like repeats that interact to form a rod-like structure(See e.g., Winkler et al., Eur. J. Biochem., 248:193 [1997]). In someembodiments, human alpha-actinin spectrin-like repeats are utilized(Genbank accession number M86406; SEQ ID NO:87; FIG. 16). In otherembodiments, alpha-actinin homologs from other organisms are utilized(e.g., mouse (Genbank accession number AJ289242); Xenopus (Genbankaccession number BE576799); and rat (Genbank accession number AF190909).

B. Variants

Still other embodiments of the present invention provide mutant orvariant forms of spectrin-like repeats (ie., muteins). It is possible tomodify the structure of a peptide having an activity of spectrin-likerepeats for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life, and/or resistance toproteolytic degradation in vivo). Such modified peptides provideadditional peptides having a desired activity of the subjectspectrin-like repeats as defined herein. A modified peptide can beproduced in which the amino acid sequence has been altered, such as byamino acid substitution, deletion, or addition.

Moreover, as described above, variant forms (e.g., mutants) of thesubject spectrin-like repeats are also contemplated as finding use inthe present invention. For example, it is contemplated that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e., conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Accordingly, some embodiments of the presentinvention provide variants of spectrin-like repeats containingconservative replacements. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (See e.g., Stryer (ed.),Biochemistry, 2nd ed, W H Freeman and Co. [1981]). Whether a change inthe amino acid sequence of a peptide results in a functional homolog canbe readily determined by assessing the ability of the variant peptide tofunction in a fashion similar to the wild-type protein. Peptides inwhich more than one replacement has taken place can readily be tested inthe same manner.

The present invention further contemplates a method of generating setsof combinatorial mutants of the present spectrin-like repeats, as wellas truncation mutants, and is especially useful for identifyingpotential variant sequences (i.e., homologs) that possess the biologicalactivity of spectrin-like repeats (e.g., a decrease in muscle necrosis).In addition, screening such combinatorial libraries is used to generate,for example, novel spectrin-like repeat homologs that possess novelbiological activities all together.

Therefore, in some embodiments of the present invention, spectrin-likerepeat homologs are engineered by the present method to produce homologswith enhanced biological activity. In other embodiments of the presentinvention, combinatorially-derived homologs are generated which providespectrin-like repeats that are easier to express and transfer to hostcells. Such spectrin-like repeats, when expressed from recombinant DNAconstructs, can be used in therapeutic embodiments of the inventiondescribed below.

Still other embodiments of the present invention provide spectrin-likerepeat homologs which have intracellular half-lives dramaticallydifferent than the corresponding wild-type protein. For example, thealtered proteins comprising the spectrin-like repeat homologs arerendered either more stable or less stable to proteolytic degradation orother cellular process that result in destruction of, or otherwiseinactivate spectrin-like repeats. Such homologs, and the genes thatencode them, can be utilized to alter the pharmaceutical activity ofconstructs expressing spectrin-like repeats by modulating the half-lifeof the protein. For instance, a short half-life can give rise to moretransient biological effects. As above, such proteins find use inpharmaceutical applications of the present invention.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population ofspectrin-like repeat homologs are aligned, preferably to promote thehighest homology possible. Such a population of variants can include,for example, spectrin-like repeat homologs from one or more species, orspectrin-like repeat homologs from different proteins of the samespecies (e.g., including, but not limited to, those described above).Amino acids that appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialspectrin-like repeat library is produced by way of a degenerate libraryof genes encoding a library of polypeptides that each include at least aportion of candidate spectrin-like repeat sequences. For example, amixture of synthetic oligonucleotides is enzymatically ligated into genesequences such that the degenerate set of candidate spectrin-like repeatsequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g., for phage display) containingthe set of spectrin-like repeat sequences therein.

There are many ways by which the library of potential spectrin-likerepeat homologs can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential spectrin-likerepeat sequences. The synthesis of degenerate oligonucleotides is wellknown in the art (See e.g., Narang, Tetrahedron Lett., 39:3 9 [1983];Itakura el al., Recombinant DNA, in Walton (ed.), Proceedings of the 3rdCleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289[1981]; Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura etal., Science 198:1056 [1984]; Ike et al. Nucl. Acid Res., 11:477[1983]). Such techniques have been employed in the directed evolution ofother proteins (See e.g., Scott et al., Science, 249:386-390 [1980];Roberts et al., Proc. Natl. Acad. Sci. USA, 89:2429-2433 [1992]; Devlinet al., Science, 249: 404-406 [1990]; Cwirla et al., Proc. Natl. Acad.Sci. USA, 87: 6378-6382 [1990]; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815, each of which is incorporated herein byreference).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques are generally adaptable for rapid screening of the genelibraries generated by the combinatorial mutagenesis of spectrin-likerepeat homologs. The most widely used techniques for screening largegene libraries typically comprise cloning the gene library intoreplicable expression vectors, transforming appropriate cells with theresulting library of vectors, and expressing the combinatorial genesunder conditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate sequences created by combinatorial mutagenesistechniques.

Accordingly, in one embodiment of the present invention, the candidategenes comprising altered spectrin-like repeats are displayed on thesurface of a cell or viral particle, and the ability of particular cellsor viral particles to bind to a another member of the DPC complex (e.g.,actin) is assayed. In other embodiments of the present invention, thegene library is cloned into the gene for a surface membrane protein of abacterial cell, and the resulting fusion protein detected by panning (WO88/06630; Fuchs et al., BioTechnol., 9:1370 [1991]; and Goward et al.,TIBS 18:136 [1992]). In other embodiments of the present invention,fluorescently labeled molecules that bind proteins comprising spectrinlike repeats (e.g., actin), can be used to score for potentiallyfunctional spectrin-like repeat homologs. Cells are visually inspectedand separated under a fluorescence microscope, or, where the morphologyof the cell permits, separated by a fluorescence-activated cell sorter.

In an alternate embodiment of the present invention, the gene library isexpressed as a fusion protein on the surface of a viral particle. Forexample, foreign peptide sequences are expressed on the surface ofinfectious phage in the filamentous phage system, thereby conferring twosignificant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of infection. The group of almostidentical E. coli filamentous phages M13, fd, and fl are most often usedin phage display libraries, as either of the phage gIII or gVIII coatproteins can be used to generate fusion proteins without disrupting theultimate packaging of the viral particle (See e.g., WO 90/02909; WO92/09690; Marks et al., J. Biol. Chem., 267:16007 [1992]; Griffths etal., EMBO J., 12:725 [1993]; Clackson et al., Nature, 352:624 [1991];and Barbas et al., Proc. Natl. Acad. Sci., 89:4457 [1992]).

In another embodiment of the present invention, the recombinant phageantibody system (e.g., RPAS, Pharmacia Catalog number 27-9400-01) ismodified for use in expressing and screening of spectrin-like repeatcombinatorial libraries. The pCANTAB 5 phagemid of the RPAS kit containsthe gene that encodes the phage gIII coat protein. In some embodimentsof the present invention, the spectrin-like repeat combinatorial genelibrary is cloned into the phagemid adjacent to the gIII signal sequencesuch that it is expressed as a gIII fusion protein. In other embodimentsof the present invention, the phagemid is used to transform competent E.coli TG1 cells after ligation. In still other embodiments of the presentinvention, transformed cells are subsequently infected with M13KO7helper phage to rescue the phagemid and its candidate spectrin-likerepeat gene insert. The resulting recombinant phage contain phagemid DNAencoding a specific candidate spectrin-like repeat and display one ormore copies of the corresponding fusion coat protein. In someembodiments of the present invention, the phage-displayed candidateproteins that are capable of, for example, binding to actin, areselected or enriched by panning. The bound phage is then isolated, andif the recombinant phage express at least one copy of the wild type gIIIcoat protein, they will retain their ability to infect E. coli. Thus,successive rounds of reinfection of E. coli and panning will greatlyenrich for spectrin-like repeat homologs, which can then be screened forfurther biological activities.

In light of the present disclosure, other forms of mutagenesis generallyapplicable will be apparent to those skilled in the art in addition tothe aforementioned rational mutagenesis based on conserved versusnon-conserved residues. For example, spectrin-like repeat homologs canbe generated and screened using, for example, alanine scanningmutagenesis and the like (Ruf et al., Biochem., 33:1565 [1994]; Wang etal., J. Biol. Chem., 269:3095 [1994]; Balint et al. Gene 137:109 [1993];Grodberg et al., Eur. J. Biochem., 218:597 [1993]; Nagashima et al., J.Biol. Chem., 268:2888 [1993]; Lowman et al., Biochem., 30:10832 [1991];and Cunningham et al., Science, 244:1081 [1989]), by linker scanningmutagenesis (Gustin et al., Virol., 193:653 [1993]; Brown et al., Mol.Cell. Biol., 12:2644 [1992]; McKnight et al., Science, 232:316); or bysaturation mutagenesis (Meyers et al., Science, 232:613 [1986]).

C. Truncations and Additions

In yet other embodiments of the present invention, the spectrin-likerepeats of human dystrophin are replaced by truncation or additions ofspectrin-like repeats from dystrophin or another protein, including, butnot limited to, those described above. Accordingly, in some embodiments,amino acids are truncated from either end of one or more of thespectrin-like repeats in a given construct. The activity of truncationmutants is determined using any suitable assay, including, but notlimited to, those disclosed herein.

In some embodiments, additional amino acids are added to either or bothends of the spectrin-like repeats in a given construct. In someembodiments, single amino acids are added and the activity of theconstruct is determined. Amino acids may be added to one or more of thespectrin-like repeats in a given construct. The activity ofspectrin-like repeats comprising additional amino acids is determinedusing any suitable assay, including, but not limited to, those disclosedherein.

III. Carboxy-Terminal Domain Truncated Dystrophin Genes

In some embodiments, the present invention provides compositionscomprising nucleic acid, wherein the nucleic acid encodes amini-dystrophin peptide, and wherein the mini-dystrophin peptidecomprises a substantially deleted dystrophin C-terminal domain (e.g.,55% of the dystrophin C-terminal domain is missing). In someembodiments, this type of truncation prevents the mini-dystrophinpeptide from binding both syntrophin and dystrobrevin.

The dystrophin COOH-terminal domain is located adjacent to thecysteine-rich domain, and contains an alternatively spliced region andtwo coiled-coil motifs (Blake et al., Trends Biochem. Sci., 20:133,1995). The alternatively spliced region binds three isoforms ofsyntrophin in muscle, while the coiled-coil motifs bind numerous membersof the dystrobrevin family (Sadoulet-Puccio et al., PNAS, 94:12413,1997). The dystrobrevins display significant homology with theCOOH-terminal region of dystrophin, and the larger dystrobrevin isoformsalso bind to the syntrophins. The importance and functional significanceof syntrophin and dystrobrevin remains largely unknown, although theymay be involved in cell signaling pathways (Grady et al., Nat. Cell.Biol, 1:215, 1999).

Researchers have previously generated transgenic mdx mouse strainsexpressing dystrophins deleted for either the syntrophin or thedystrobrevin binding domain (Rafael et al., Hum. Mol. Genet., 3:1725,1994; and Rafael et al., J. Cell Biol., 134:93 1996). These micedisplayed normal muscle function and essentially normal localization ofsyntrophin, dystrobrevin, and nNOS. Thus, while dystrobrevin appears toprotect muscle from damage (Grady et al., Nat. Cell. Biol, 1:215, 1999),removal of the dystrobrevin binding site from dystrophin does not resultin a dystrophy. Subsequent studies revealed that syntrophin anddystrobrevin bind each other in addition to dystrophin, so that removalof only one of the two binding sites on dystrophin might not sever thelink between dystrophin, syntrophin and dystrobrevin. Surprisingly, thetransgenic mice according to the present invention (See Example 1)displayed normal muscle function even though they lacked both thesyntrophin and dystrobrevin binding sites.

IV. MCK Regulatory Regions

In certain embodiments, nucleic acid encoding mini-dystrophin peptidesof the present invention are operably linked to muscle creatine kinasegene (MCK) regulatory regions and control elements, as well as mutatedfrom of these regions and elements (see See U.S. Provisional App. SerNo. 60/218,436, filed Jul. 14, 2000, and International ApplicationPCT/US01/22092, filed Jul. 13, 2001, both of which are herebyincorporated by reference). In some embodiments, the nucleic acidencoding mini-dystrophin peptides is operably linked to these sequencesto provide muscle specificity and reduced size such that the resultingconstruct is able to fit into, for example, a viral vector (e.g.adeno-associated virus). MCK gene regulatory regions (e.g. promoters andenhancers) display striated muscle-specific activity and have beencharacterized in vitro and in vivo. The major known regulatory regionsin the mouse MCK gene include a 206 base pair muscle-specific enhancerlocated approximately 1.1 kb 5′ of the transcription start site in mouse(i.e. SEQ ID NO:87) and a 358 base pair proximal promoter (i.e. SEQ IDNO:93) [Shield, et al., Mol. Cell. Biol., 16:5058 (1996)]. A larger MCKpromoter region may also be employed (e.g. SEQ ID NO:92), as well assmaller MCK promoter regions (e.g. SEQ ID NO:94).

The 206 base pair MCK enhancer (SEQ ID NO:87) contains a number ofsequence motifs, including two classes of E-boxes (MCK-L and MCK-R),CarG, and AT-rich sites. Similar E-box sequences are found in theenhancers of the human, rat, and rabbit MCK genes [See, Trask, et al.,Nucleic Acids Res., 20:2313 (1992)]. Mutation may be made to thissequence by, for example, inserting an additional MCK-R control elementinto a wild-type enhancer sequence naturally containing one MCK-Rcontrol element (such that the resulting sequence has at least two MCK-Rcontrol elements). For example, the inserted MCK-R control elementreplaces the endogenous MCK-L control element. The 206 base pair mouseenhancer (SEQ ID NO:2) may be modified by replacing the left E-box(MCK-L) with a right E-Box (MCK-R) to generate a mutant muscle-specificenhancer region (e.g. to generate SEQ ID NO:88). A similar approximately200 base pair wild type enhancer region in human may be modified byreplacing the left E-box with a MCK-R to generate a mutantmuscle-specific enhancer region (e.g. 2R human enhancer regions).

Another modification that may be made to generate mutant muscle-specificenhancer regions by inserting the S5 sequence GAGCGGTTA (SEQ ID NO:95)into wild type mouse, human, and rat enhancer sequence. Making such amodification to the mouse enhancer SEQ ID NO:87, for example, generatesS5 mutant muscle-specific enhancer regions (e.g. SEQ ID NO:89). Anothermodification that may be made, for example, to the wild type mouseenhancer is replacing the left E-box (MCK-L) with a right E-Box (MCK-R),and also inserting the 5S sequence, to generate 2R5S type sequences (e.gin mouse, SEQ ID NO:90). These mutant muscle-specific enhancer regionsmay have additional sequences added to them or sequences that are takenaway. For example, the mutant muscle-specific enhancer regions may havea portion of the sequence removed (e.g. the 3′ 41 base pairs). Examplesof such mutant truncation 2RS5 sequences in mouse is SEQ ID NO:91 withthe 3′ 41 base pairs removed, generating mutant truncated 2RS5muscle-specific enhancer regions.

Any of these wild-type or mutant muscle-specific enhancer regionsdescribed above may be further modified to produce additional mutants.These additional mutants include, but are not limited to,muscle-specific enhancer regions having deletions, insertions orsubstitutions of different nucleotides or nucleotide analogs so long asthe transcriptional activity of the enhancer region is maintained.Guidance in determining which and how many nucleotide bases may besubstituted, inserted or deleted without abolishing the transcriptionalactivity may be found using computer programs well known in the art, forexample, DNAStar software or GCG (Univ. of Wisconsin) or may bedetermined empirically using assays provided by the present invention.

V. Expression Vectors

The present invention contemplates the use of expression vectors withthe compositions and methods of the present invention (e.g. with thenucleic acid constructs encoding the mini-dystrophin peptides). Vectorssuitable for use with the methods and compositions of the presentinvention, for example, should be able to adequately package and carrythe compositions and cassettes described herein. A number of suitablevectors are known in the art including, but are not limited to, thefollowing: 1) Adenoviral Vectors; 2) Second Generation AdenoviralVectors; 3) Gutted Adenoviral Vectors; 4) Adeno-Associated VirusVectors; and 5) Lentiviral Vectors.

Those skilled in the art will recognize and appreciate that othervectors are suitable for use with methods and compositions of thepresent invention. Indeed, the present invention is not intended to belimited to the use of the recited vectors, as such, alternative meansfor delivering the compositions of the present invention arecontemplated. For example, in various embodiments, the compositions ofthe present invention are associated with retrovirus vectors and herpesvirus vectors, plasmids, cosmids, artificial yeast chromosomes,mechanical, electrical, and chemical transfection methods, and the like.Exemplary delivery approaches are discussed below.

1. Adenoviral Vectors

Self-propagating adenovirus (Ad) vectors have been extensively utilizedto deliver foreign genes to a great variety of cell types in vitro andin vivo. “Self-propagating viruses” are those which can be produced bytransfection of a single piece of DNA (the recombinant viral genome)into a single packaging cell line to produce infectious virus;self-propagating viruses do not require the use of helper virus forpropagation. As with many vectors, adenoviral vectors have limitationson the amount of heterologous nucleic acid they are capable ofdelivering to cells. For example, the capacity of adenovirus isapproximately 8-10 kb, the capacity of adeno-associated virus isapproximately 4.8 kb, and the capacity of lentivirus is approximately8.9 kb. Thus, the mutants of the present invention that provide shorternucleic acid sequences encoding the mini-dystrophin peptides (comparedto full length wild-type dystrophin (14 kb)), improve the carryingcapacity of such vectors.

2. Second Generation Adenoviral Vectors

In an effort to address the viral replication problems associated withfirst generation Ad vectors, so called “second generation” Ad vectorshave been developed. Second generation Ad vectors delete the earlyregions of the Ad genome (E2A, E2B, and E4). Highly modified secondgeneration Ad vectors are less likely to generate replication-competentvirus during large-scale vector preparation, and complete inhabitationof Ad genome replication should abolish late gene replication. Hostimmune response against late viral proteins is thus reduced [SeeAmalfitano et al., “Production and Characterization of ImprovedAdenovirus Vectors With the E1, E2b, and E3 Genes Deleted,” J. Virol.72:926-933 (1998)]. The elimination of E2A, E2B, and E4 genes from theAd genome also provide increased cloning capacity. The deletion of twoor more of these genes from the Ad genome allows for example, thedelivery of full length or cDNA dystrophin genes via Ad vectors[Kumar-Singh et al, Hum. Mol. Genet., 5:913 (1996)].

3. Gutted Adenoviral Vectors

“Gutted,” or helper dependent, Ad vectors contain cis-acting DNAsequences that direct adenoviral replication and packaging but do notcontain viral coding sequences [See Fisher et al. “RecombinantAdenovirus Deleted of All Viral Genes for Gene Therapy of CysticFibrosis,” Virology 217:11-22 (1996) and Kochanek et al. “A NewAdenoviral Vector: Replacement of All Viral Coding Sequences With 28 kbof DNA Independently Expressing Both Full-length Dystrophin andBeta-galactosidase′” Proc. Nat. Acad. Sci. USA 93:5731-5736 (1996)].Gutted vectors are defective viruses produced by replication in thepresence of a helper virus, which provides all of the necessary viralproteins in trans. Since gutted vectors do not contain any viral genes,expression of viral proteins is not possible.

Recent developments have advanced the field of gutted vector production[See Hardy et al., “Construction of Adenovirus Vectors Through Cre-loxRecombination,” J. Virol. 71:1842-1849 (1997) and Hartigan-O'Conner etal., “Improved Production of Gutted Adenovirus in Cells ExpressingAdenovirus Preterminal Protein and DNA Polymerase,” J. Virol.73:7835-7841 (1999)]. Gutted Ad vectors are able to maximallyaccommodate up to about 37 kb of exogenous DNA, however, 28-30 kb ismore typical. For example, a gutted Ad vector can accommodate the fulllength dystrophin or cDNA, but also expression cassettes or modulatorproteins.

4. Adeno-Associated Virus Vectors

In preferred embodiments, the nucleic acid encoding the mini-dystrophinpeptides of the present invention are inserted in adeno-associatedvectors (AAV vectors). AAV vectors evade a host's immune response andachieve persistent gene expression through avoidance of the antigenicpresentation by the host's professional APCs such as dendritic cells.Most AAV genomes in muscle tissue are present in the form of largecircular multimers. AAV's are only able to carry about 5 kb of exogenousDNA. As such, the nucleic acid of the present invention encoding themini-dystrophin peptides is well suited, in some embodiments, forinsertion into these vectors due the reduced size of the nucleic acidsequences.

The dystrophin expression cassettes of the present invention (containingnucleic acid encoding mini-dystrophin peptides) may be cloned into anyof a variety of cis-acting plasmid vectors that contain theadeno-associated virus inverted terminal repeats (ITRs) to allowproduction of infectious virus. For example, one such plasmid is thecis-acting plasmid (pCisAV) (Yan et al., PNAS, 97:6716-6721, 2000). Thisplasmid contains the AAV-ITRs separated by a NotI cloning site. The ITRelements were derived from pSub201, a recombinant plasmid from which aninfectious adeno-associated virus genome can be excised in vitro andused to study viral replication. After ligation of the dystrophinexpression cassette (isolated as a NotI fragment frompCK6DysR4-23-71-78An) into NotI-digested pCisAV, rAAV stocks aregenerated by cotransfection of pCisAV. CK6DysR4-23-71-78An and pRep/Cap(Fisher, et al., J. Virol. 70:520-532, 1996) together with coinfectionof the recombinant adenovirus Ad.CMVlacZ into 293 cells. Recombinant AAVvector, for example, may then be purified on CsCl gradients as described(Duan, et al., Virus Res. 48:41-56, 1997).

5. Lentiviral Vectors

Vectors based on human or feline lentiviruses have emerged as anothervector useful for gene therapy applications. Lentivirus-based vectorsinfect nondividing cells as part of their normal life cycles, and areproduced by expression of a package-able vector construct in a cell linethat expresses viral proteins. The small size of lentiviral particlesconstrains the amount of exogenous DNA they are able to carry to about10 kb. However, once again, the small size nucleic acid encoding themini-dystrophin peptides of the present invention allow such vectors tobe employed.

6. Retroviruses

Vectors based on Moloney murine leukemia viruses (MMLV) and otherretroviruses have emerged as useful for gene therapy applications. Thesevectors stably transduce actively dividing cells as part of their normallife cycles, and integrate into host cell chromosomes. Retroviruses maybe employed with the compositions of the present invention (e.g. genetherapy), for example, in the context of infection and transduction ofmuscle precursor cells such as myoblasts, satellite cells, or othermuscle stem cells.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); ° C. (degrees Centigrade); and Sigma (Sigma Chemical Co.,St. Louis, Mo.).

EXAMPLE 1 Carboxy-Terminal Domain Truncated Dystrophin Genes

This example describes the generation of carboxy-terminal truncateddystrophin nucleic acid sequences. In particular, this examplesdescribes the construction of dystrophin nucleic acid sequence with theentire carboxy-terminal domain deleted, and testing of this sequence ina mouse model for DMD.

A. Methods

The bases encoding amino acids 3402-3675 (corresponding to exons 71-78)were deleted from the full length murine dystrophin cDNA (SEQ ID NO:2,accession No. M68859) by recombinant PCR, leaving the last three aminoacids (exon 79) of the dystrophin protein unaltered. This dystrophinΔ71-78 cDNA was cloned into an expression vector containing bases −2139to +239 of the human-skeletal actin (HSA) promoter (Brennan, et al. J.Biol. Chem. 268:719, 1993). A splice acceptor from the SV40 VP1 intron(isolated as a 400 bp HindIII/XbaI fragment from pSVL; AmershamPharmacia Biotech) was inserted immediately 3′ of the HSA fragment, andthe SV40 polyadenylation signal (isolated as a BamHI fragment frompCMVβ; MacGregor and Caskey, Nuc. Acid. Res., 17:2365, 1989) wasinserted 3′ of the dystrophin cDNA. The excised dystrophin Δ71-78expression cassette was injected into wild-type C57B1/10×SJL/J F2 hybridembryos, and F_(o) mice were screened by PCR. Five positive F_(o)'s werebackcrossed onto the C57B1/10mdx background, and the line with the mostuniform expression levels was selected for analysis. Also employed werepreviously described transgenic mdx mice that express dystrophinconstructs deleted approximately for exons 71-74 (Δ71-74) or exons 75-78(Δ75-78), which remove amino acids 3402-3511 and 3528-3675,respectively, See Rafael et al., J. Cell Biol., 134:93-102, 1996).Transgenic mdx line Dp71 expresses the Dp71 isoform of dystrophin instriated muscle (Cox et al., Nat. Genet., 8:333-339, 1994).

i. Morphology Methods

Quadriceps, soleus, extensor digitorum longus (EDL), tibialis anterior,and diaphragm muscles were removed from the mice, frozen in liquidnitrogen cooled O.C.T. embedding medium (Tissue-Tek), and cut into 7-μmsections. After fixing in 3.7% formaldehyde, sections were stained inhematoxylin and eosin-phloxine. Stained sections were imaged with aNikon E1000 microscope connected to a Spot-2 CCD camera. To determinethe percentage of fibers containing central nuclei, the number of musclefibers with centrally-located nuclei was divided by the total number ofmuscle fibers.

ii. Evans Blue Assays

4 month old control mice and Δ71-78 mice were analyzed after injectionwith Evans blue, as described previously (Straub et al., J. Cell. Biol.,139:375-385, 1997). In brief, mice were tail vein-injected with 150 μlof a solution containing 10 mg/ml Evans blue dye in PBS (150 mM NaCl, 50mM Tris, pH 7.4). After 3 hours, the animals were euthanized and mousetissues were either fixed in 3.7% formaldehyde/0.5% glutaraldehyde toobserve gross dye uptake, or frozen unfixed in O.C.T. embedding medium.To examine Evans blue uptake by individual fibers, 7-μm-thick frozensections were fixed in cold acetone and analyzed by fluorescencemicroscopy.

iii. Immunofluorescence Assays

Quadriceps and diaphragm muscles from C57B1/10, mdx, and Δ71-78 micewere removed, frozen in O.C.T. embedding medium, and cut into 7-μmsections. Immunofluorescence was performed with previously describedantibodies against dystrophin (NH₂ terminus), α1-syntrophin (SYN17),β1-syntrophin, α-dystrobrevin-1 (DB670), α-dystrobrevin-2 (DB2), andutrophin. After incubation with primary antibodies, cryosections wereincubated with an FITC-conjugated goat anti-rabbit secondary antibodyand fluorescent images were viewed on a Nikon E1000 microscope.Antibodies to α-sarcoglycan (Rabbit 98), β-sarcoglycan (Goat 26),γ-sarcoglycan (Rabbit 245), δ-sarcoglycan (Rabbit 215), sarcospan(Rabbit 235), α-dystroglycan (Goat 20), β-dystroglycan (AP 83), or nNOS(Rabbit 200) have been described previously (Duclos e al., J. Cell.Biol., 142:1461, 1998). Cy3-conjugated secondary antibodies were usedand images were viewed on a Bio-Rad MRC-600 laser scanning confocalmicroscope. All digitized images were captured under the sameconditions.

iv. Measurements of Contractile Properties Methods

Contractile properties of muscles from 6-month-old Δ71-78 transgenicmice were compared with those of C57B1/10 wild-type and mdx mice usingmethods described previously (Lynch et al., Am. J. Physiol., 272:C2063,1997). The samples included eight muscles each from the EDL, soleus, anddiaphragm. Mice were deeply anesthetized with avertin and each musclewas isolated and dissected free from the mouse. After removal of thelimb muscles, the mice were euthanized with the removal of the diaphragmmuscle. The muscles were immersed in a bath filled with oxygenatedbuffered mammalian Ringer's solution (137 mM NaCl, 24 mM NaHCO₃, 11 mMglucose, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, and 0.025 mMtubocurarine chloride, pH 7.4). For each muscle, one tendon was tied toa servomotor and the other tendon to a force transducer. Muscles werestretched from slack length to the optimal length for force developmentand then stimulated at a frequency that produced absolute isometrictetanic force (mN). After the measurements of the contractileproperties, the muscles were removed from the bath, blotted and weighedto determine muscle mass. Specific force (kN/m²) was calculated bydividing absolute force by total fiber cross sectional area.

v. Muscle Membrane Isolation Methods

Muscle microsomes from 12-14 month-old C57B1/10, mdx, Δ71-78, Δ71-74,Δ75-78, and Dp71 mice were prepared as described previously (Ohlendiecket al., J. Cell. Biol., 112:135, 1991). In brief, skeletal muscle washomogenized in 7.5-vol homogenization buffer plus protease inhibitorComplete (Boehringer). The homogenate was centrifuged at 14,000 g for 15min to remove cellular debris. The supernatant was filtered throughcheesecloth and spun at 142,000 g for 37 minutes to collect microsomes.The microsome pellet was resuspended in KCl wash buffer (0.6 M KCl, 0.3M sucrose, 50 mM Tris-HCl, pH 7.4) plus protease inhibitors andrecentrifuged at 142,000 g for 37 minutes to obtain KCl-washedmicrosomes. The final pellet was resuspended in 0.3 M sucrose and 20 mMTris-maleate, pH 7.0. Samples were quantified by the Coomassie PlusProtein Assay Reagent (Pierce Chemical Co.) and equivalent proteinloading was verified by SDS-PAGE. KCl-washed microsomes were analyzed byWestern blot using antibodies against β2-syntrophin, pan syntrophin,nNOS (Transduction Laboratories), β-dystroglycan, α-sarcoglycan(Novocastra Laboratories), and other proteins described above.

B. Results

i. Generation of Dystrophin Δ71-78 Transgenic Mice

To test the function of a dystrophin protein lacking both the syntrophinand dystrobrevin binding sites, we prepared a cDNA expression vectordeleted for the COOH-terminal domain (corresponding to exons 71-78; SeeFIG. 19) as described above. The structure of several dystrophintransgenic constructs previously tested are also shown for comparison.Mice expressing the dystrophin Δ71-78 transgene were crossed onto themdx background and dystrophin levels were analyzed by Western blotting.The expression of the dystrophin Δ71-78 transgene in skeletal muscle wasdetermined to be 10-fold higher than endogenous dystrophin.Immunofluorescent staining of quadriceps muscle using an antibodyagainst the NH₂-terminus of dystrophin revealed that the Δ71-78 proteinwas localized to the sarcolemma, similar to wild-type dystrophin.Dystrophin Δ71-78 expression was also found to be uniform in thediaphragm, EDL, and soleus muscles, but the tibialis anterior muscledisplayed a mosaic expression pattern. The human skeletal muscle-actinpromoter used in this study was not expressed in either smooth orcardiac muscle.

ii. Morphology of Dystrophin Δ71-78 Mice Appears Normal

We initially analyzed transgenic mdx mouse muscle tissues formorphological signs of dystrophy. Hematoxylin and eosin-stained limb anddiaphragm skeletal muscle sections of dystrophin Δ71-78 mice revealednone of the signs of fibrosis, necrotic fibers, or mononuclear cellinfiltration that were apparent in age-matched mdx controls. NMJs(neuromuscular junctions) of transgenic mice stained withrhodamine-labeled -bungarotoxin consistently appeared normal in contrastto the varying degrees of postsynaptic folding observed in mdx NMJs. Mdxmuscle fibers have previously been shown to be highly permeable to thevital dye Evans blue in vivo, reflecting damage to the dystrophic fibersarcolemma (Matsuda et al., J. Biochem. (Tokyo), 118:959, 1995).Skeletal muscle fibers from dystrophin Δ71-78 mice, like wild-typeanimals, were found not to be permeable to Evans blue dye.

iii. Analysis of Centrally Nucleated Muscle Fibers

Another hallmark of dystrophy in mdx mice is the presence of largenumbers of centrally-nucleated muscle fibers, reflecting cycles of fiberdegeneration and regeneration (Torres and Duchen, Brain, 110:269, 1987).To estimate the degree of myofiber regeneration occurring in Δ71-78transgenic mice, centrally nucleated fibers were counted from a varietyof muscle groups in age-matched wild-type, mdx, and Δ71-78 mice (See,Table 2). By 4 months of age, 71% of muscle fibers in mdx quadricepsmuscles contained central nuclei, whereas wild-type muscles had <1%.Interestingly, 4 month old dystrophin Δ71-78 quadriceps musclesdisplayed 1% central nuclei, indicating that very little, if any,regeneration was occurring. When 1-year-old mice were compared, a modestincrease in centrally nucleated fibers became apparent. Quadricepsmuscles from Δ71-78 mice contained 10% centrally nucleated fibers,although diaphragm muscles still displayed <1%. EDL and soleus musclesdisplayed 5 and 8% centrally nucleated fibers, respectively. Forcomparison, 1-year-old wild-type mice had <1% centrally nucleated fibersin both limb and diaphragm muscles. Furthermore, 1-year-old mdx limbmuscles had 60% centrally nucleated fibers, whereas the diaphragm had35%.

TABLE 2 Percentage of Centrally Nucleated Fibers in Mouse SkeletalMuscles Line Age Quad Dia TA EDL Soleus C57/B110 4 <1 <1 ND ND ND mdx 471 58 ND ND ND Δ71-78 4 1 <1 ND ND ND C57/B110 12 <1 <1 <1 <1 <1 mdx 1265 35 58 50 61 Δ71-78 12 10 <1 ND 5 8 Δ71-74 15 5 <1 <1 <1 ND Δ75-78 158 <1 4 2 7 Quad = quadriceps; Dia = diaphragm; TA = tibialis anterior;Age is in months

Previous studies of transgenic mice expressing dystrophins deleted forexons Δ71-74 (Δ71-74) or exons Δ75-78 (Δ75-78) revealed no increase inthe numbers of centrally nucleated fibers by 4 months of age (Rafael etal. 1996, see above). To contrast these mice with the 71-78 transgenics,central nuclei counts were performed on 15-month-old Δ71-74 and 75-78mice. It was determined that these animals had central nuclei counts inbetween those of wild-type and Δ71-78 mice. The Δ71-74 and Δ75-78 micehad 5 and 8% centrally nucleated fibers in quadriceps, respectively(Table 2).

iv. Contractile Properties

Compared with muscles of wild-type mice, those from mdx mice displayed asignificant amount of necrosis, fibrosis, and infiltrating mononuclearcells. mdx skeletal muscles also displayed a loss of specificforce-generating capacities when muscles were stimulated to contract invitro, providing an extremely sensitive and quantitative measurement ofthe dystrophic process (FIG. 20A). In contrast, dystrophin Δ71-78 micehad no major abnormalities when subjected to the same analysis (FIG.20B). Muscle mass for both EDL and diaphragm were not significantlydifferent between dystrophin Δ71-78 and wild-type mice, whereasdystrophin Δ71-78 soleus muscles were slightly hypertrophied. Whenstimulated to contract, all three muscle groups displayed specificforces not significantly different from wild-type (P<0.05). Theseresults demonstrate that the dystrophin Δ71-78 protein has essentiallythe same functional capacity as the full-length protein.

V. Localization of the DAP Complex in Δ71-78 Mice

Immunofluorescent analysis of the peripheral DAP complex revealedα1-syntrophin, β1-syntrophin, α-dystrobrevin-1, and α-dystrobrevin-2 tobe localized at the sarcolemma with dystrophin, despite the lack ofsyntrophin and dystrobrevin binding sites in the transgene-encodeddystrophin. α1-syntrophin levels were similar between wild-type andΔ71-78 mice. However, the levels of β1-syntrophin were elevated at themembrane in Δ71-78 mice, particularly in those fibers that normallyexpress significant levels of this isoform. α-dystrobrevin-1 wasprimarily located at the NMJ in wild-type mice, and was exclusivelylocated at the NMJs in mdx mice. Surprisingly, in dystrophin Δ71-78mice, higher levels of α-dystrobrevin-1 were observed at the sarcolemmathan in wild-type mice. The Δ71-78 mice also displayed a slight increasein utrophin localization along the sarcolemma, but this increase wasless than the increase in mdx fibers. Immunofluorescent localization ofthe sarcoglycans, α- and β-dystroglycan, sarcospan, and nNOS in Δ71-78mice revealed no differences in the expression of these proteins whencompared with wild-type mice. The proper localization of these proteinsto the sarcolemma indicated that membrane targeting of the DAP complexcomponents can proceed in the absence of the COOH-terminal domain ofdystrophin.

vi. DAP Complex Protein Levels

To examine the levels of the DAP complex members that associate withdystrophin, muscle microsomes were prepared from wild-type anddystrophin Δ71-78 mice and analyzed by Western blotting. This approachprovides information on the relative abundance of individual DAP complexmembers in muscles of separate lines of mice. Slightly elevated levelsof β-dystroglycan were detected in dystrophin Δ71-78 mice, which we havepreviously observed whenever dystrophin is overexpressed. Isoforms ofsyntrophin and dystrobrevin were present at slightly different levelswhen the dystrophin Δ71-78 membranes were compared with those fromwild-type mice. α1-syntrophin and β2-syntrophin levels were lower thanin wild-type mice, whereas the level of β1-syntrophin was elevated.Although there was approximately the same amount of α-dystrobrevin-2,there were elevated levels of α-dystrobrevin-1 in Δ71-78 microsomes. Areduction in nNOS was observed in dystrophin Δ71-78 muscle, indicatingthat nNOS binds weakly to the DAP complex in Δ71-78 mice. Levels ofα-sarcoglycan were similar in all lines tested, and provided an internalcontrol for protein loading.

Since some DAP complex members exhibited isoform changes in Δ71-78 mice,we examined purified microsomes from dystrophin Δ71-74 and Δ75-78 mice.Transgenic mdx mice that express the dystrophin isoform Dp71 in musclewere also included in this study since these dystrophic mice have theDAP complex present at the sarcolemma. α1-syntrophin levels were lowerin all four transgenic lines compared with wild-type mice. Surprisingly,β1-syntrophin was absent in Δ71-74 microsomes but was highlyoverexpressed in Δ75-78 and Dp71 microsomes. The Δ71-74 microsomes hadequivalent β2-syntrophin levels when compared with wild-type microsomes,but this isoform of syntrophin was reduced in both Δ75-78 and Dp71microsomes. A pan syntrophin antibody, which detects all three isoformsof syntrophin, confirmed the upregulation of syntrophin in Δ75-78 andDp71 microsomes. Similar to Δ71-78, α-dystrobrevin-1 was elevated in alldystrophin transgenic microsome preparations. However, in comparisonwith wild-type, α-dystrobrevin-2 was higher in Δ71-74 and Δ75-78, butequal in Dp71 microsomes. Contrary to the Δ71-78 mice, deleting eitherexons 71-74 or 75-78 restored nNOS to wild-type levels. However, Dp71mice, which lack the NH₂-terminal and rod domains of dystrophin, did notretain nNOS in the microsome fractions. Previous studies have also shownthat utrophin is upregulated in mdx and Dp71 mice (Ohlendieck et al.,Neuron, 7:499-508, 1991). Therefore, utrophin levels were compared inall transgenic lines and we found that Δ71-78, Δ71-74, and Δ75-78 micedo not have the elevated levels seen in mdx and Dp71 mice.

EXAMPLE 2 Construction of ΔR4-R23, ΔR2-R21+H3, and ΔR2-R1

This example describes the construction of R4-R23 (micro-dys1),ΔR2-R21+H3 (micro-dys3), and ΔR2-R1 (micro-dys2), three sequences with 4spectrin-like repeat encoding sequences. The ‘full-length’ humandystrophin cDNA that was started with was actually a sequence slightlysmaller than the true full-length human dystrophin cDNA. In particular,the starting sequence, called full-length HDMD (SEQ ID NO:47, see FIG.23) is the same as the wild-type human dystrophin in SEQ ID NO:1, exceptthe 3′ 1861 base pairs are deleted (at an XbaI site), and the 39 basepair alternatively spliced exon 71 (bases 10432-10470) are deleted. Thissequence (SEQ ID NO:47) is originally in pBSX (SEQ ID NO:46, See FIGS.21 and 22).

A. Cloning ΔR4-R23

The procedure used for cloning ΔR4-R23 is outlined in FIG. 24.Initially, three PCR reactions were performed (employing Pfu polymerase)to create the deletion shown in FIG. 24. The primers employed in thefirst reaction were 5′ GAA CAA GAT TCA CAC AAC TGG C 3′ (SEQ ID NO:48),which anneals to 1954-1975 of the HDMD clone, and 5′ GTT CCT GGA GTC TTTCAA GAT CCA CAG TAA TCT GCC TC 3′ (SEQ ID NO:49), which is a reversedtailed primer (the bold sequence anneals to 2359-2341 of the HDMD clone,and the underlined sequence anneals to 9023-9005 the HDMD clone. PCR wasconducted employing these primers, and a 425 bp PCR product wasproduced. The first primer employed in the second reaction was 5′ GAGGCA GAT TAC TGT GGA TCT TGA AAG ACT CCA GGA AC 3′ (SEQ ID NO:50), whichis the reverse complement primer of SEQ ID NO:49 (the bold-facedsequence of SEQ ID NO:50) anneals to 2341-2359 of the HDMD clone in theforward direction. The underlined sequence anneals to 9005-9023 of theHDMD clone in the forward direction. The other primer employed for thesecond reaction was 5′ TGT TTG GCG AGA TGG CTC 3′ (SEQ ID NO:51) whichanneals to 9413-9396 of HDMD in the reverse direction. PCR was conductedemploying these primers, and a 427 bp PCR product was produced. Thethird reaction employed the products from steps 1 and 2 and the outsideprimers SEQ ID NO:48 and SEQ ID NO:51, producing a 814 bp fragment byPCR. This fragment was then digested with NcoI and HindIII to produce a581 bp DNA fragment.

This 581 bp fragment was then cloned into a 5016 bp NcoI+Hind IIIfragment from the HDMD clone. The 581 bp fragment contained part ofrepeat 3, all of Hinge 2, and part of repeat 24. The NcoI site used inthe HDMD clone was located at 2055 bp. The Hind III site was located at9281 bp. The 5016 fragment contained the pBSX cloning vector sequence,and the entire 5′ UTR, the entire N terminus, Hinge 1, Repeats 1, 2, andpart of repeat 3 up to the NcoI site of human dystrophin. Ligation ofthe 5016 bp fragment and 581 bp fragment (step 2) was then performed tocreated a 5597 bp sequence.

Step 3 was then performed to clone a 2.9 kb HindIII fragment containingpart of repeat 24, the C terminus, and the 3′ UTR (See FIG. 24). The 5′HindIII site is located at 9281 bp of the HDMD clone. The 3′ HindIIIsite of this fragment is derived from pBSX polylinker. This 2.9 kbfragment was cloned into the HindIII site of the product of Step 2 toyield an 8.5 kb plasmid, composed of the ΔR4-R23 cDNA plus pBSX. Theentire ΔR4-R23 cDNA was excised from pBSX with NotI and cloned into theNotI site of the HSA expression vector (HSA promoter—VP1 intron—NotIsite—tandem SV40 poly adenylation site).

B. Cloning ΔR2-R21+H3

The procedure used for cloning ΔR2-R21+H3 is outlined in FIG. 25.Initially, four PCR reactions were performed (employing Pfu polymerase)to create the deletion shown in FIG. 25. The primers employed in thefirst reaction were 5′ GAT GTG GAA GTG GTG AAA GAC 3 (SEQ ID NO:52),which anneals to 1319-1330 of the HDMD clone, and 5′ CCA ATA GTG GTC AGTCCA GGA GCA TGT AAA TTG CTT TG 3′ (SEQ ID NO:53), which is a reverse,tailed primer (the bold-faced sequence anneals to 1546-1532 of the HDMDclone and the underlined sequence anneals to 7512-7490 of the HDMDclone. PCR was conducted with these primers and a 228 bp PCR product wasproduced. The first primer employed in the second reaction was 5′ CAAAGC AAT TTA CAT GCT CCT GGA CTG ACC ACT ATT GG 3′ (SEQ ID NO:54), whichis the reverse complement of SEQ ID NO:53 (the bold-faced sequence ofSEQ ID NO:54 anneals to 1532-1546 of the HDMD clone in the forwarddirection, and the underlined sequence anneals to 7512-7490 of the HDMDclone in the forward direction. The other primer employed in the secondreaction was 5′ CTG TTG CAG TAA TCT ATG CTC CAA CAT CAA GGA AGA TG 3′(SEQ ID NO:55), and the bold-faced sequence anneals to 8287-8270 of theHDMD clone, and the underlined sequence anneals to 7612-7593 of the HDMDclone as a reverse primer. PCR was performed with these primers, and a123 bp PCR product was produced. The first primer employed in the thirdreaction was 5′ CAT CTT CCT TGA TGT TGG AGC ATA GAT TAC TGC AAC AG 3′(SEQ ID NO:56), the underlined sequence anneals to 7593-7612 of the HDMDclone in the forward direction, and the bold-faced sequence anneals to8270-8287. The second primer employed in the third reaction was SEQ IDNO:51 (see above), which anneals to 9413-9396 in the reverse direction.PCR was performed with these primers, and a 1143 bp fragment wasproduced. The fourth reaction employed the products from reactions 1, 2,and 3 as template, and the outside primers (SEQ ID NO:52 and SEQ IDNO:51), and a 1494 bp fragment was produced using Pfu polymerase.

This 1494 bp fragment was then digested with MunI and HindIII to producea 1270 bp band and cloned into a 4320 bp MunI+HindIII fragment from theHDMD clone. The 1270 bp fragment contained the part of repeat 1, all ofhinge 3, repeat 22, repeat 23, and part of repeat 24. The 4320 bpfragment contained the 5′ UTR of HDMD, the N terminus, Hinge 1, and partof repeat 1 and pBSX. The MunI site in HDMD is located at base 1409. TheHindIII site is at 9281 bp. Ligation of the 4320 bp fragment and the1270 bp fragment was then performed (See FIG. 25) and a 4490 bp fragmentwas produced. Step 3 was performed as describe above for ΔR4-R23 togenerate ΔR2-R21+H3.

C. Cloning ΔR2-R21

The cloning of ΔR2-R21 was performed essentially the same way as forΔR2-R21+H3, with the exception of the recombinant PCR reaction toassemble the rod domain deletion (See, FIG. 26). All other steps are thesame. Three PCR reactions were performed (using Pfu polymerase) tocreate the deletion. The primers employed in the first reaction were SEQID NO:52 (see above), and 5′ CTG TTG CAG TAA TCT ATG ATG TAA ATT GCT TTG3′ (SEQ ID NO:57), the underlined sequence anneals to 8287-8270 of theHDMD clone in the reverse direction, and the bold-faced sequence annealsto 1546-1532 of the HDMD clone in the reverse direction. PCR wasperformed with these primers, and a 250 bp product was obtained. Thefirst primer employed in the second reaction was 5′ CAA AGC AAT TTA CATCAT AGA TTA CTG CAA CAG 3′ (SEQ ID NO:58), which is is the reversecomplement of SEQ ID NO:57 (the bold-faced sequence of SEQ ID NO:58anneals to 1532-1546 of the HDMD clone in the forward direction, and theunderlined sequence anneals to 8270-8287 of the HDMD clone in theforward direction. The other primer employed in the second reaction wasSEQ ID NO:51, which anneals to 9413-9396 in the reverse direction. PCRwas performed with these primers and a 1143 bp product was obtained. Thethird reaction employed the products from reactions 1 and 2 (astemplate) and the outside primers (SEQ ID NO:52 and SEQ ID NO:51), and a1383 bp fragment was produced. This fragment was then digested with MunIand HindIII to produce an 1147 bp fragment containing part of repeat 1,repeat 22, repeat 23, and part of repeat 24. This was then cloned intothe same MunI+HindIII HDMD fragment described for the ΔR2-R21+H3 cloneand all other steps thereafter were the same.

EXAMPLE 3 ΔR4-R23 Deletions

This example describes the construction of 5′ UTR, 3′ UTR, andC-terminal deletions of ΔR4-R23 (making it even smaller), as well as theaddition of polyadenylation and promoter sequences. This example alsodescribes the alteration of the Kozak sequence (to become more like thatof consensus).

A. Deletion of the 3′ UTR

In order to delete the 3′ UTR, the following two primers were employed5′ TCT CTC CAA GAT CAC CTC G 3′ (SEQ ID NO:64), which anneals to9117-9134 of the HDMD full length clone, and 5′ ATG AAG CTT GCG GCC GCATGC GGG AAT CAG GAG TTG 3′ (SEQ ID NO:65) (the underlined site is aHindIII site that was included in this primer and the bold-faced type isa NotI site). SEQ ID NO:65 is a reverse primer that anneals to11340-11322 of HDMD in the 3′ UTR. These primers cause the deletion of707 bp of the 3′ UTR from the XbaI cloning site located at 12057 to theend of this primer (SEQ ID NO:65), leaving 113 bp of native 3′ UTR, andintroducing NotI and HindIII cloning sites. The PCR product obtainedusing the primers corresponding to SEQ ID NOS:64 and 65 on the pΔR4-R23clone was named HdysΔ3′UTR and was saved for use as a template togenerate a further deletion of exons 71-78 (see part C below).

B. Deletion of 5′ UTR and Alteration of Kozak Sequence

A portion of the 5′ UTR was deleted (and the Kozak sequence was alteredin the same step). The ‘step 2’ clone from cloning of ΔR4-R23 wasutilized (this was the the product of ligating the step 1 PCR productinto the 5016 bp NcoI and HindIII fragment from the HDMD full-lengthclone, and this clone contained pBSX backbone plus the 5′ UTR, Nterminus, Hinge 1, Repeats 1, 2, 3, Hinge 2, and part of repeat 24.There is an MunI site located in the first repeat at nucleotide 1409 ofthe HDMD cDNA. In addition, there is a NotI site that is polylinkerderived at the 5′ end of the clone. These two sites were employed,MunI+NotI, to clone a new fragment containing a truncated 5′ UTR and analtered Kozak sequence as follows. PCR was performed, using Pfupolymerase using the following primers. The first primer was 5′ TAG CGGCCG CGG TTT TTT TTA TCG CTG CCT TGA TAT ACA CTT TCC ACC ATG CTT TGG TGGGAA GAA GTA G 3′ (SEQ ID NO:59). We created a NotI site (underlined) inthis primer so the product could be cloned back into the NotI site fromthe polylinker. The sequence immediately 3′ to this NotI sitecorresponds to the dystrophin 5′ UTR sequence (the original Kozaksequence was changed with this primer, from TCAAAATGC, changed toCCACCATGC. The second primer was 5′ TTT TCC TGT TCC AAT CAG C 3′ (SEQ IDNO:60) which anneals to sequence 1441-1423 of HDMD full length clone.The final product of this reaction was 1270 bp and was digested withNotI+MunI to produce a 1233 bp fragment that was then cloned into theNotI (polylinker)+MunI site in Repeat 1 of the “Step 2” clones(described above for ΔR4-23). This new clone was named pHDMD5′ Kozak.

C. Deletion of Exons 71-78 (C-terminal)

Using three PCR reactions, a 71-78 deletion was created. We used theHindIII fragment containing the 3′UTR that was generated by digestion ofthe HDMD full-length dystrophin cDNA with HindIII as the vector to clonethe 71-78 fragment into the HindIII site. The primer employed for thefirst reaction were 5′ GGC TTC CTA CAT TGT GTC AGT TTC CAT GTT GTC CCC3′ (SEQ ID NO:66), and 5′ TCT CTC CAA GAT CAC CTC 3′ (SEQ ID NO:67)anneals to 9117-9134 of HDMD. PCR was performed employing these primersand a 1334 bp product was produced. The primers for the second reactionwere SEQ ID NO:65, and 5′ GGG GAC AAC ATG GAA ACT GAC ACA ATG TAG GAAGCC 3′ (SEQ ID NO:68), where the bold-face sequence anneals to exon 70at 10415-10431 in the forward direction, and the underlined sequenceanneals to 11216-11233 in the forward direction. PCR was performed and a150 bp fragment was generated. The product of reactions 1 and 2 wereused as template and the outside primers SEQ ID NO:65 and SEQ ID NO:67were used to prime the reaction which generated the complete 71-78 Cterminus (1484 bp). This product was digested with HindIII to produce a1319 bp fragment and was cloned into the HindIII site of pTZ19R (SeeFIG. 35). This new clone was named pTZ-HDMD-H3Δ71-78Δ3.

D. Cloning of the SV40 pA Sequence into the Not I Site

The next step was the cloning of the SV 40 pA sequence:

(SEQ ID NO:71) 5′GATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTCG GATC3′into the NotI site of the 3′ HindIII fragment that now contains the 3′UTR and 71-78. A PCR reaction was performed on the template pHSA with areverse primer 5′ AGC GGC CGC AAA AAA CCT CCC ACA CCT CC 3′ (SEQ IDNO:69, containing a regenerating NotI site—underlined) and 5′ TAC GGCCGA TCC AGA CAT GAT AAG ATA C 3′ (SEQ ID NO:70, containing a destroyingEagI site, in bold). All other sequence (besides the NotI and EagIsites) is SV40 pA. This PCR reaction generated a 195 bp product+cloningsites=209 bp. We then cloned this fragment into the NotI site ofpTZ-HDMD-H3Δ71-78Δ3 generated by PCR in the 3′ UTR clone. The upstream(5′-most) NotI site in this clone was destroyed by EagI ligation. Thisnew clone was named pTZ-HDMD-H33′A.

E. Cloning of CK6 Promoter into NotI Site

The CK6 promoter—

(SEQ ID NO:61) 5′GGT- ACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGGCCCAATCAAGGCTGTGGGGGACTGAGGGCAGGCTGTAACAGGCTTGGGGGCCAGGGCTTATACGTGCCTGGGACTCCCAAAGTATTACTGTTCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCCCGGGTCACGGGGATCCTCTAGACC-3′was amplified using two tailed primers: 5′ AGC GGC CGC GGT ACT ACG GGTCTA GG 3′ Forward (SEQ ID NO:62), and 5′ ATC GGC CGT CTA GAG GAT CCC CGTGAC C 3′ Reverse (SEQ ID NO:63). The underlined sequence is a NotI siteadded to the end of this primer. The remaining sequence is CK6 sequence.The bold-faced type is an EagI site added to the end of this primer. Theremaining sequence is from CK6. The CK6 promoter was amplified this wayso we could add the NotI and EagI sites (so the entire cassette could beexcised when put back together with NotI). This PCR product wastherefore digested with NotI and EagI and ligated into the NotI site ofpHDMD5′Kozak. This new clone was named pCK6HDMD5′Kozak. NotI and EagIproduce compatible cohesive sites, but when EagI ligates to NotI, itdestroys the site. So we placed the EagI site at the 3′ end, so thatwhen the final construct was cut with NotI, the entire expressioncassette could be excised intact. The same strategy was employed at the3′ end when placing the SV40 poly A sequence into the 3′ Not I site.

F. Re-ligating the 5′ and 3′ Ends.

This step was performed as described above in the micro-dystrophintransgene constructs. We reconstituted the same cloning sites but withmodifications in the fragments, so the modified 3′ end, isolated as aHindIII fragment from clone pTZ-HDMD-H33′A (example 3 part D), was ableto be cloned into the HindIII site of pCK6HDMD5′Kozak (example 3, partE). This final clone, named pCK6R4-R23KozakΔ3′, contains a truncateddystrophin expression cassette that can be excised in its entirety bydigestion with NotI. This excised expression cassette can then be usedfor a variety of purposes. One such purpose is to clone the cassetteinto a plasmid containing the inverted terminal repeats fromadeno-associated virus. By cloning the dystrophin expression cassetteHDMD-H33′A into a cloning site between the two ITRs of AAV, arecombinant AAV vector could be produced.

EXAMPLE 4 Construction of Reduced Repeat Dystrophin Constructs

This example describes the construction of ΔH2-R19 (an 8spectrin-like-repeat sequence), pΔR9R16 (a 16 spectrin-like-repeatsequence), pΔR1R24 (a zero spectrin-like-repeat sequence), pΔH2-H3 (an 8spectrin-like repeat sequence), and ΔH2-R19,R20 (a 7 spectrin-likerepeat sequence). One starting plasmid was pHBMD, a human dystrophincDNA (full-length HDMD, SEQ ID NO:47) with a further deletion of thesequences encoded by exons 17-48. The cDNA was cloned into thecommercially available plasmid vector pTZ19r (MBI Fermentas; Genbankaccession number Y14835, See FIG. 35), into which an EcoRI-SalI adapter(prepared by self-annealing of the oligonucleotide 5′-AATTCGTCGACG-3′,SEQ ID NO:83) had been ligated into the the EcoRI site. Base number 1 ofthe cDNA is immediately 3′ of the adapter sequence, and the cDNA ends atthe XbaI site at base 12,100 of SEQ ID NO:1. This XbaI site had beenligated into the XbaI site of the plasmid ptZ19r. Another startingplasmid is pBSX (SEQ ID NO:46), a modified version of pBluescript KSII+(Stratagene) which is used to make pBSXA (pBSX into which the SV40polyadenylation signal (pA) was added). This pA sequence was excised asa 206 bp fragment from pCMVβ (Clonetech), blunt-ended with DNApolymerase I, and ligated into the blunt-ended KpnI site of pBSX.

Another starting plasmid is pCK3, which is pBSX with the 3.3 kb mousemuscle creatine kinase enhancer plus promoter attached to the minxintron (See, Hauser et al., Mol Ther., 2:16-25, 2000). Another staringplasmid is pHDSK, which is pHBMD digested with KpnI, to remove thedystrophin sequences 3′ of the internal KpnI site (base 7,616 of thehuman dystrophin cDNA sequence, SEQ ID NO:1). A further starting vectoris p44.1, which is pBluescript KS− (Stratagene) carrying a humandystrophin cDNA fragment spanning the EcoRI site at base 7,002 to theEcoRI site at base 7,875 of the full-length human dystrophin cDNAsequence, cloned into the EcoRI site of the vector. Another plasmidemployed was p30-2, pBluescribe (Stratagene) containing a fragment fromthe full-length human dystrophin cDNA spanning bases 1,455 to the EcoRIsite at base 2,647, cloned into the EcoRI site of the vector. Anadditional vector employed was p30-1, pBluescribe (Stratagene)containing an EcoRI fragment from the full-length human dystrophin cDNAspanning bases 2,647 to 4,558, cloned into the EcoRI site of the vector.An further plasmid employed is p47-4, pBluescript KS− (Stratagene)carrying the human dystrophin cDNA EcoRI fragment spanning bases 4,452to 7,002 of the full-length cDNA sequence, cloned into the EcoRI site ofthe vector. Another plasmid is p9-7, pBluescribe (Stratagene) containingbases 1-1,538 of the full-length human dystrophin cDNA. Base 1 isattached to a linker of the sequence 5′ GAATTC-3′ and cloned into theEcoRI site of the vector. Base 1,538 is blunt-end cloned into the PstIsite of the vector, which had been destroyed by fill-in with T4 DNApolymerase. Another vector employed is p63-1, pBluescript KS−(Stratagene) carrying the human dystrophin cDNA EcoRI fragment spanningbases 7,875 to the 3′ end of the full-length cDNA, cloned into the EcoRIsite of the vector (the 3′ end of the cDNA is ligated to a linker of thesequence 5′-GAATTC-3′).

Initially, the MCK promoter plus enhancer and the minx intron wereexcised from pCK3 by digestion with EagI, yielding a 3.5 kb fragmentthat was ligated into EagI-digested pBSXA to make pBSXACK3. Truncateddystrophin cDNAs, derived from pHBMD, containing various deletions ofdystrophin domains were prepared as described below. The cDNA insertswere excised from the plasmid backbone with SalI, and ligated intopBSXACK3 at the SalI site, which is located between the minx intron andthe pA sequence, such that the 3′ end of the cDNA was adjacent to the pAsequence. The isolation of the truncated cDNAs is described below.pBSXACK3-truncated dystrophin plasmids were digested with BssHII torelease the expression vectors, which were gel purified and used togenerate transgenic mice.

Isolation of ΔH2R19

A PCR product was generated by amplification of plasmid p30-2 withprimers

(SEQ ID NO:72) 5′-TGTGCTGCAAGGCGATTAAGTTGG-3′ and (SEQ ID NO:75)5′-GAGCTAGGTCAGGCTGCTGTGAAATCTGTGC-3′.Primers SEQ ID NO:75 overlaps the end of repeat 3 and the beginning ofhinge 3. Primer SEQ ID NO:72 corresponds to a sequence in the plasmidvector adjacent to the cloning site. A second PCR product was generatedby amplification of plasmid p44-1 using primers5′-CCAGGCTTTACACTTTATGCTTCC-3′ (SEQ ID NO:73) and5′-GCACAGATTTCACAGCAGCCTGACCTAGCTC-3′ (SEQ ID NO:74). Primer SEQ IDNO:74 is the reverse complement of primer SEQ ID NO:75. Primer SEQ IDNO:73 corresponds to a sequence in the plasmid vector adjacent to thecloning site. The PCR products were then purified by agarose gelelectorphoreses, and quantified. A recombinant PCR product was thenprepared by mixing together 10 ng of each of the first two PCR products,then re-PCR amplifying using only primers SEQ ID NO:72 and SEQ ID NO:73.This recombinant PCR product was then digested with NheI and KpnI, andligated into NheI and KpnI digested pHΔSK to generate plasmid pHBMDΔH2(NheI cuts at cDNA base 1,519, and KpnI cuts at base 7,616 of thefull-length human dystrophin cDNA sequence). pHBMDΔH2 was then digestedwith KpnI and XbaI, and ligated to the KpnI-XbaI fragment from pHBMD(this latter fragment contains the full-length human dystrophin cDNAbases 7,616 to 12,100) to obtain plasmid pΔH2R19.

Isolation of pΔR9R16

Plasmid p44-1 was digested with EcoRI and Asp718 to excise a 610 bp cDNAinsert, that was ligated into pBSX digested with EcoRI and Asp718,yielding pBSX44AE. pBSX44AE was digested with EcoRI and XbaI, andligated to the NheI-EcoRI cDNA-containing fragment from p30-2, yieldingpBSX44AE/30-2NE. Plasmid pBSX44AE/30-2NE was linearized by digestionwith EcoRI, into which was ligated the EcoRI-digested recombinant PCRproduct ΔR9-R16. This latter recombinant PCR product was generated asfollows. Plasmid p30-1 was amplified with primers SEQ ID NO:72 and5′-CCATTTCTCAACAGATCTTCCAAAGTCTTG-3′ (SEQ ID NO:77), and plasmid p47-4was amplified by PCR with primers SEQ ID NO:73 and5′-CAAGACTTTGGAAGATCTGTTGAGAAATGG-3 (SEQ ID NO:76). A recombinant PCRproduct (ΔR9-R16) was then prepared by mixing together 10 ng of each ofthe first two PCR products, then re-PCR amplifying using only primersSEQ ID NO:72 and SEQ ID NO:73. This recombinant PCR product was thendigested with EcoRI, and ligated into EcoRI digested pBSX44AE/30-2NE togenerate plasmid pR9R16int. Plasmid pR9R16int was digested with NcoI andAsp718, and the 3 kb cDNA fragment was isolated and ligated into NcoIand Asp718 digested pHΔSK to generate pΔR9R16.

Isolation of pΔR1R24

Plasmid p9-7 was PCR amplified with PCR primers 5′-AGTGTGGTTTGCCAGCAGTC(SEQ ID NO:80) and 5′-CAAAGTCCCTGTGGGCGTCTTCAGGAGCTTCC-3′ (SEQ IDNO:79). Plasmid p63-1 was PCR amplified with primers 5′GGAAGCTCCTGAAGACGCCCACAGGGACTTTG-3′ (SEQ ID NO:78) and5′-TGGTTGATATAGTAGGGCAC-3′ (SEQ ID NO:81). A recombinant PCR product(ΔR1-R24) was then prepared by mixing together 10 ng of each of thefirst two PCR products, then re-PCR amplifying using only primers SEQ IDNO:80 and SEQ ID NO:81. This recombinant PCR product was then digestedwith SexAI and PpuMI, and ligated into SexAI and PpuMI digested pHBMD togenerate plasmid pΔR1R24.

Isolation of pΔH2-H3

This clone was prepared exactly as pΔH2-R19, except that primer5′-CAGATTTCACAGGCTGCTCTGGCAGATTTC-3′ (SEQ ID NO:82) was used in place ofprimer SEQ ID NO:74, and primer 5′-GAAATCTGCCAGAGCAGCCTGTGAAATCTG-3′(SEQ ID NO:84) was used in place of primer SEQ ID NO:75.

Isolation of ΔH2-R19,R20

This clone was generated from clone pΔH2R19 as follows. Plasmid p44-1was amplified with primers SEQ ID NO:72 and5′-TGAATCCTTTAACATAGGTACCTCCAACAT-3′ (SEQ ID NO:85). Plasmid 63-1 wasamplified with primers 5′-ATGTTGGAGGTACCTATGTTAAAGGATTCA-3′ (SEQ IDNO:86) and SEQ ID NO:81. The PCR products were then purified by agarosegel electorphoreses, and quantified. A recombinant PCR product was thenprepared by mixing together 10 ng of each of the first two PCR products,then re-PCR amplifying using only primers SEQ ID NO:72 and SEQ ID NO:81.This product was digested with Asp718 and BstXI, and ligated into Asp718and BstXI digested pHBMD generating clone pBMDΔR20. The Asp718-XbaIcDNA-containing fragment from pBMDΔR20 was isolated and ligated intoAsp718 and XbaI digested pΔH2R19 to generate pΔH2-R19,R20.

EXAMPLE 5 Testing Truncated Dystrophin in mdx Mice

This example describes the generation of transgenic mdx mice expressingtruncated dystrophin cDNA (see above), and testing these mice in variousways to determine various measurable muscle values. A variety ofdystrophin expression cassettes (FIG. 27) were used to generatetransgenic mice to test their functional capacity in alleviatingmuscular dystrophy on the dystrophin null mdx background. FIG. 27depicts the truncated dystrophin cDNA sequences tested, all of whichwere linked to an regulatory regions, a minx intron, and the SV40polyadenylation sequence (the 4-repeat constructs employed the HSA actinpromoter, See Crawford et al., J. Cell. Biol., 150:1399, 2000; and theremaining sequences employed an MCK enhancer and promoter, see Niwa etal., Genes Dev. 4:1552, 1990). Each of these constructs was released bydigestion from plasmid hosts, were gel purified, and used to generatetransgenic mice.

Excised expression cassettes injected into wild type C57B1/10×SJL/J F2hybrid embryos, and F⁰ mice were screened by PCR analysis of DNAisolated from tail snips. Positive F⁰ mice were backcrossed onto theC57B1/10mdx background, and individual mouse lines were tested fordystrophin expression by immunofluorescent analysis with dystrophinantibodies for of expression in skeletal muscle fibers. Lines thatdisplayed uniform expression of dystrophin in muscle fibers wereselected for further analysis. These lines were further backcrossed ontothe mdx mouse background before analysis of dystrophin expression,muscle function and morphology.

A. Truncated Dystrophin cDNAs are Expressed at Various Levels in Musclesof Transgenic mdx Mice.

Muscle extracts were analyzed by western (immuno) blot analysis todetermine the amount of dystrophin made in different muscles of thetransgenic mdx mice. For these studies, total protein was extracted fromthe quadriceps and diaphragm muscles of control and transgenic mice, andprotein concentrations were determined using the Coomassie Plus ProteinAssay Reagent (Pierce). One hundred micrograms of each sample waselectrophoresed on a 6% polyacrylamidc/SDS gel (29.7:0.3/acryl:bis),transferred for 2 hours at 75 volts onto Biotrace Nitrocellulose (GelmanScience) in 1× Tris-Glycine, 20% methanol, 0.05% SDS, using awet-transfer apparatus (Hoefer). Membranes were blocked in 10% non-fatdry milk, 1% normal goat serum, and 0.1% Tween-20, and hybridized withDYS1 (Novacastra) at a 1/1000 dilution for 2 hours at room temperature,washed, and then probed with horse radish peroxidase conjugatedanti-mouse antibodies at a 1/2,000 dilution (Cappel). Blots weredeveloped using the ECL chemiluminescence system (Amersham). Allincubations contained 1% normal goat serum and 0.1% Tween-20. Theresults of the western blot indicated that R9-R16 was poorly expressedin this line of mice, especially in the diaphragm, and that H2-H3 wasvery poorly expressed in the diaphragm.

B. Truncated Dystrophin cDNAs Confer Various Degrees of Protection onMuscles of Transgenic mdx Mice.

Various muscle groups from the different lines of transgenic miceexpressing truncated dystrophins were examined for morphologicalabnormalities, and for expression of dystrophin by indirectimmunofluorescence (IF) in individual fibers. IF analysis was performedas follows. Skeletal muscle was removed from control and transgenicanimals, cut into strips, embedded in Tissue-tek OCT mounting media(Miles, Inc.), and frozen quickly in liquid nitrogen-cooled isopentane.Seven micrometer sections were blocked with 1% gelatin in KPBS for 15minutes, washed in KPBS+0.2% gelatin (KPBSG), and incubated for 2 hoursin KPBSG+1% normal goat serum with affinity-purified dystrophin antibody18-4 (Cox et al., Nature, 364:725-729, 1993) at a dilution of 1/1000.After washing, the slides were incubated for 1 hour with eitherbiotin-labeled goat anti-rabbit polyclonal antibodies (Pierce), washedagain, and incubated with FITC (fluorescein isothiocynate)-conjugatedstreptavidin. After a final wash, Vectashield (Vector Laboratories,Inc.) with DAPI was applied and sections were photographed through adual bandpass filter under 40× magnification using a Nikon E1000microscope.

Morphological analysis of the muscles was performed as follows. Musclegroups from among the following types were chosen for analysis:Quadriceps (Quad), soleus, extensor digitorum longus (EDL), tibialisanterior (TA), and diaphragm muscles. Selected muscles were removed frommice, frozen in liquid nitrogen cooled O.C.T. embedding medium(Tissue-Tek), and cut into 7 μm sections. After fixing in 3.7%formaldehyde, sections were stained in hematoxylin and eosin-phloxine.Stained sections were imaged with a Nikon E1000 microscope andphotographed.

The results of this analysis show that micro-dystrophin expression(ΔR4R23 transgene) in the diaphragm prevents the onset of musculardystrophy in mdx mice. In particular, micro-dystrophin transgenic andwild-type C57B1/10 diaphragm sections stained with hematoxylin and eosin(H&E) show morphologically healthy muscle without areas of fibrosis,necrosis, mononuclear cell infiltration, or centrally located nuclei.Conversely, the mdx diaphragm displays a high level of dystrophicmorphology by H&E. Also, immuno-fluorescence, using anti-dystrophinpolyclonal primary antisera, demonstrates that micro-dystrophintransgenes are expressed at the sarcolemmal membrane in a similarfashion to that of wild-type dystrophin, while mdx mice do not expressdystrophin.

H & E staining also shows that truncated dystrophins with 8 or 16spectrin-like repeats have varying abilities to prevent dystrophy in thediaphragm of transgenic mdx mice. The H2R19 maintains normal musclemorphology that is not different from wild-type C57B1/10 muscle. TheΔH2R19 muscle displays a very low percentage of centrally nucleatedfibers, while the ΔH2-R19,R20 and ΔR9-16 constructs display percentagesintermediate between ΔH2-R19 and mdx (see FIG. 28). The mdx diaphragmhad a large number of centrally nucleated fibers, many necrotic fibers,and large areas of mono-nuclear cell infiltration and fibrosis.

The results also show that quadriceps muscle fibers expressingmicro-dystrophin transgene (ΔR4R23 transgene) display normal morphologyand exclude Evans Blue Dye. Micro-dystrophin transgenic mdx or C57B1/10quadriceps sections stained with hematoxylin and eosin (H&E) displaymorphologically healthy muscle without areas of necrosis, fibrosis,mononuclear cell infiltration, or centrally-located nuclei, as opposedto sections of mdx muscle. The high abundance of central nuclei andmononuclear immune cell infiltration are evidence of muscle cellnecrosis. Imrnunofluorescence results indicate that micro-dystrophinsdisplay a subsarcolemmal expression pattern like that of wild-typedystrophin, while mdx mice do not express dystrophin. Evans Blue Dye(EBD) uptake is an indication of a damaged myofiber. For analysis of EBDuptake, mice were tail vein injected with 150 μl of a solutioncontaining 10 mg/ml Evans blue dye in PBS (150 mM NaCl, 50 mM Tris pH7.4). After three hours, the animals were euthanized and mouse tissueswere either fixed in 3.7% formaldehyde/0.5% glutaraldehyde to observegross dye uptake, or frozen unfixed in O.C.T. embedding medium. Toexamine Evans blue uptake by individual fibers, 7 μm thick frozensections were fixed in cold acetone and analyzed by fluorescencemicroscopy. The results of this testing indicate that fibers expressingmicro-dystrophin or wild-type dystrophin exclude EBD, and that damagedmdx muscle cell membranes are permeable to Evans Blue dye.

A hallmark of dystrophy in mdx mice is the presence of large numbers ofcentrally-nucleated muscle fibers, reflecting cycles of fiberdegeneration and regeneration. To estimate the degree of myofiberregeneration occurring in the transgenic mice, centrally-nucleatedfibers were counted from quadriceps muscles in age-matched wild-type,mdx, and transgenic mdx mice (FIG. 28). To determine the percentage offibers containing central nuclei, the number of muscle fibers withcentrally-located nuclei was divided by the total number of musclefibers.

Expression of 8 or 4 repeat micro-dystrophin transgenes on the mdxbackground significantly reduces the percentage of fibers withcentrally-located nuclei to wild-type or near wild-type levels (FIG.28). Dystrophin molecules with zero repeats are unable to correct themdx phenotype by this assay. The best constructs were observed to be the8 repeat H2-R19 and the 4 repeat R2-R23 constructs. Greater percentagesof centrally nucleated fibers were observed in mice expression the exon17-48 deletion, the 4 repeat R2R21 construct, the 7 repeat H2R19,R20construct, the 16 repeat R9R16 construct, and the zero repeat R1R24construct (FIG. 28). The results from the R9R16 construct likely do notreflect the full functional capacity of the 16 repeat dystrophin sincethis line of mice expressed very low levels of the truncated dystrophinprotein. All other muscles expressed levels of dystrophin that have beenshown to be capable of preventing dystrophy if the expressed protein isfunctional (Phelps et al., Hum Mol Genet; 4:1251-1258, 1995).

The functional capacity of the truncated dystrophins was also assessedby measuring muscle contractile properties in the transgenic mdx mice.Contractile properties of muscles from transgenic mice were comparedwith those of C57B1/10 wild type and mdx mice. The samples included 4-8muscles each from the tibialis anterior (TA), extensor digitorum longus(EDL) or diaphragm. Mice were deeply anesthetized with avertin and eachmuscle was isolated and dissected free from the mouse. After removal ofthe limb muscles, the mice were euthanized with the removal of thediaphragm muscle. The muscles were immersed in a bath filled withoxygenated buffered mammalian Ringer's solution (137 mM NaCl, 24 mMNaHCO₃, 11 mM glucose, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄,and 0.025 mM tubocurarine chloride, pH 7.4). For each muscle, one tendonwas tied to a servomotor and the other tendon to a force transducer.Muscles were stretched from slack length to the optimal length for forcedevelopment and then stimulated at a frequency that produced absoluteisometric tetanic force (mN). Following the measurements of thecontractile properties, the muscles were removed from the bath, blottedand weighed to determine muscle mass. Specific force (kN/m2) wascalculated by dividing absolute force by total fiber cross sectionalarea.

FIG. 29 shows that the 8 repeat dystrophin encoded by H2-R19 supportsnormal force development in both the diaphragm (FIG. 29 a) and EDLmuscle (FIG. 29 b). In contrast, previous studies showed that the exon17-48 construct, which encodes a dystrophin with 8.25 spectrin-likerepeats, supports only 90-95% of normal force development in thediaphragm (Phelps et al., Hum Mol Genet, 4:1251-1258, 1995). The 8repeat dystrophin lacking a central hinge (H2-H3), and tile 7 repeatdystrophin (H2-R19,R20) both fail to support significant forcegeneration compared with dystrophic mdx muscles. The results from theR9-R16 construct likely do not reflect the full functional capacity ofthe 16 repeat dystrophin, since this line of mice expressed very lowlevels of the truncated dystrophin.

FIG. 30 shows that the micro-dystrophin transgenic mdx mice develop lessspecific force than do C57B1/10 mice in the TA, but near wild-typelevels in the diaphragm. Micro-dys 1 and −2 refer to transgenes ΔR4-R23,and ΔR2-R21, respectively. FIG. 30A shows that C57B1/10 mice displaysignificantly higher specific force than both transgenic lines and mdxmice in the tibialis anterior (TA) muscle. Data are presented as means±standard error of the means (s.e.m.) with each bar representing 6 to 8TA muscles. ANOVA statistical testing was performed. (* indicatessignificance from C57B1/10, p<0.01; s indicates significance fromC57B1/10, p<0.05). FIG. 30B shows that mice expressing Micro-dys 1develop wild type levels of specific force in the diaphragm, while miceexpressing Micro-dys 2 develop ˜22% less specific force by the sameassay when compared with C57B1/10. Both lines of mice develop morespecific force than mdx mice in the diaphragm. Data are presented as thepercentage of wild type.

Dystrophic mice are susceptible to contraction-induced injury (Petrof,et al., Proc. Natl. Acad. Sci. USA. 90:3710-3714, 1993). In this part ofthe example tested whether the 4 repeat dystrophin clones would protectmuscles of transgenic mdx mice from contraction induced injuries. Totest contraction-induced injury, an experimental protocol consisting oftwo muscle stretches was performed in live, anesthetized animals. Thedistal tendon of the TA was cut and secured to the lever arm of aservomotor that monitors position and force produced by the muscle.Stimulation voltage and optimal muscle length (L₀) for force productionwere determined. The muscle was maximally stimulated and then stretched40% greater than L₀ (LC1) for 300 milliseconds. A second lengtheningcontraction was performed 10 seconds later (LC2). The maximum force thatthe muscle was able to produce after each stretch was measured andexpressed as a percentage of the force produced before stretch. Mdx miceexpressing micro-dystrophins were significantly protected from thedramatic force deficit produced after a lengthening contraction comparedwith mdx mice (FIG. 31). Micro-dys 1 and -2 refer to transgenes ΔR4-R23,and ΔR2-R21, respectively. Furthermore, there was no significantdifference between either micro-dystrophin construct studied in thisassay and C57B1/10 mice following the second, most damaging lengtheningcontraction. Data are presented as means±s.e.m. with each barrepresenting between 6 and 8 TA muscles from 9-11 week old mice.

C. Truncated 4 Repeat Dystrophin cDNAs Restore the Ability to Run LongDistances to mdx Mice.

We have observed that mdx mice are not able to run for long distances ona treadmill, as compared to wild-type mice (see below). Therefore, miceexpressing four repeat dystrophins were compared with wild-type and mdxmice for ability to run for extended times on a treadmill. Theexercising protocol utilized a six lane, enclosed treadmill with a shockgrid to allow forced running at a controlled rate. C57B1/10, C57B1/6×SJLF1, mdx or transgenic mdx mice were run at a 15 degree downward angle toinduce damaging eccentric muscle contractions. Mice were given a 15minute acclimation period prior to exercise, and then ran at 10meters/minute with a subsequent 5 m/min increase in rate every 10minutes until exhaustion. Exhaustion was determined to be the time atwhich a mouse spent more than 5 seconds sitting on the shock gridwithout attempting a re-entry to the treadmill. As shown in FIG. 32,both lines of four repeat transgenic mice ran significantly farther thanmdx mice. Micro-dys 1 and -2 refer to transgenes ΔR4-R23, and ΔR2-R21,respectively. Micro-dystrophin transgenic mice are a genetic mixture ofC57B1/6×SJL, and C57B1/10 strains, and ran an intermediate distancebetween the two wild-type lines. Data are presented as means ±s.e.m.ANOVA statistical analyses were performed. (* indicates valuessignificantly different from mdx line, p<0.01; s indicates valuessignificantly different from mdx line, p<0.05).

D. Micro-dystrophin Transgenic mdx Mice do not Display Hypertrophy

As a way to measure the functional capacity of the four-repeatdystrophins, we weighed both whole mice and dissected tibialis anteriormuscles from age matched transgenic and control mice. The results shownin FIG. 33 show that the micro-dystrophin transgenic mdx mice do notdisplay the muscle hypertrophy normally observed in mdx mice. FIG. 33Ashows that three month old micro-dystrophin transgenic mdx mice weighedsignificantly less than age-matched mdx control mice. FIG. 11B showsthat tibialis anterior (TA) muscle masses in mdx mice were significantlyhigher than control muscle masses in C57B1/10 and in both lines of mdxmice expressing different micro-dystrophin transgenes. Data arepresented as means ±s.e.m. with each bar representing between 3 and 4mice. ANOVA statistical analyses were performed (* indicates differencefrom mdx line, p<0.01; Y indicates difference from C57B1/10 line,p<0.01; s indicates difference from C57B1/10 line, p<0.05). Micro-dys 1and -2 refer to transgenes ΔR4-R23, and ΔR2-R21, respectively.

EXAMPLE 6 Mini-dystrophin-containing Adeno-associated Viral Vectors

This example describes a construct that could be made in order to allowadeno-associated virus to express a mini-dystrophin peptide in a targetmuscle cells. FIG. 34 shows a schematic illustration of a plasmid vectorcontaining the adeno-associated virus inverted terminal repeats(AAV-ITRs), the muscle promoter plus enhancer fragment known as CK6 (SEQID NO:61, the ΔR2-R21 four repeat dystrophin cDNA (SEQ ID NO:40) with afurther deletion of sequences encoded on exons 71-78, plus a 195 basepair SV40 polyadenylation signal that would have a total insert size ofapproximately 4.7 kb. The cloning capacity of adeno-associated viralvectors is approximately 4.9 kb. As such, the construct could beefficiently packaged into AAV viral particles (e.g. this plasmidconstruct could be used to transfect cells such that AAV expressingmini-dystrophin peptide is expressed). These AAV then, for example, maybe administered to a subject with DMD or BMD (i.e. gene therapy tocorrect a muscle deficiency in a subject).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmaterial science, chemistry, and molecular biology or related fields areintended to be within the scope of the following claims.

1. A composition comprising nucleic acid encoding a mini-dystrophinpeptide, wherein said mini-dystrophin peptide comprises a spectrin-likerepeat domain comprising 4 dystrophin spectrin-like repeats, whereinsaid mini-dystrophin peptide contains no more than 4 dystrophinspectrin-like repeats.
 2. The composition of claim 1, wherein saiddystrophin spectrin-like repeats are human dystrophin spectrin-likerepeats.
 3. The composition of claim 1, wherein saidmini-dystrophin-peptide is capable of increasing a measurable musclevalue in a DMD animal model by at least 20% of the wild type value,wherein said measurable muscle value is a diaphragm specific forcevalue.
 4. The composition of claim 3, wherein said mini-dystrophinpeptide is capable of increasing said diaphragm specific force value ina DMD animal model by at least 30% of the wild-type value.
 5. Thecomposition of claim 1, wherein said nucleic acid comprises anexpression vector.
 6. The composition of claim 1, wherein said nucleicacid comprises spectrin-like repeat encoding sequences.
 7. Thecomposition of claim 6, wherein said spectrin-like repeat encodingsequences are precise spectrin-like repeat encoding sequences.
 8. Thecomposition of claim 1, wherein said nucleic acid comprises anactin-binding domain encoding sequence.
 9. The composition of claim 8,wherein said actin binding domain comprises at least a portion of SEQ IDNO:6.
 10. The composition of claim 1, wherein said nucleic acidcomprises a β-dystroglycan binding domain.
 11. The composition of claim10, wherein said β-dystroglycan binding domain comprises at least aportion of a dystrophin hinge 4 encoding sequence, and at least aportion of a dystrophin cysteine-rich domain encoding sequence.
 12. Thecomposition of claim 6, wherein said spectrin-like repeat encodingsequences are selected from the group consisting of SEQ ID NOS:8-10,12-27, and 29-33.
 13. The composition of claim 1, wherein said nucleicacid contains less than 75% of a wild type dystrophin 5′ untranslatedregion.
 14. The composition of claim 1, wherein said mini-dystrophinpeptide further comprises a substantially deleted dystrophin C-terminaldomain.
 15. The composition of claim 1, wherein said nucleic acidcontains less than 50% of a dystrophin 3′ untranslated region.
 16. Acomposition comprising nucleic acid encoding a mini-dystrophin peptide,wherein said mini-dystrophin peptide comprises i) a spectrin-like repeatdomain comprising 4 dystrophin spectrin-like repeats, ii) anactin-binding domain, and iii) a β-dystroglycan binding domain; andwherein said mini-dystrophin peptide contains no more than 4 dystrophinspectrin-like repeats.
 17. The composition of claim 16, wherein saidmini-dystrophin-peptide is capable of altering increasing a measurablemuscle value in a DIVID animal model by at least 20% of the wild typevalue wherein said measurable muscle value is a diaphragm specific forcevalue.
 18. The composition of claim 17, wherein said mini-dystrophinpeptide is capable of increasing said diaphragm specific force value ina DMD animal model by at least 30% of the wild-type value.
 19. Thecomposition of claim 16, wherein said nucleic acid is less than 5.0 kbin length.
 20. A composition comprising nucleic acid encoding amim-dystrophin peptide, wherein said mini-dystrophin peptide comprises aspectrin-like repeat domain comprising 8 dystrophin spectrin-likerepeats, wherein said mini-dystrophin peptide contains no more than 8dystrophin spectrin-like repeats.
 21. The composition of claim 20,wherein said dystrophin spectrin-like repeats are human dystrophinspectrin-like repeats.
 22. The composition of claim 20, wherein saidmini-dystrophin-peptide is capable of altering a measurable muscle valuein a DMD animal model by at least 20% of the wild type value.
 23. Thecomposition of claim 16, wherein said nucleic acid contains less than50% of a dystrophin 3+ untranslated region.
 24. The composition of claim1, wherein said mini-dystrophin peptide further comprises dystrophinhinge region 1 and dystrophin hinge region
 4. 25. The composition ofclaim 24, wherein said mini-dystrophin further comprises dystrophinhinge region 2 or dystrophin hinge region
 3. 26. The composition ofclaim 16, wherein said mini-dystrophin peptide further comprisesdystrophin hinge region 1 and dystrophin hinge region
 4. 27. Thecomposition of claim 26, wherein said mini-dystrophin further comprisesdystrophin hinge region 2 or dystrophin hinge region
 3. 28. Thecomposition of claim 1, wherein said nucleic acid is less than 5.0 kb inlength.
 29. The composition of claim 5, wherein said expression vectorcomprises an adeno-associated viral sequence, and wherein said nucleicacid comprises a promoter.
 30. The composition of claim 29, wherein saidpromoter is an MCK promoter.
 31. The composition of claim 16, whereinsaid nucleic acid comprises an adeno-associated viral sequence and apromoter.
 32. The composition of claim 31, wherein said promotercomprises an MCK promoter.
 33. The composition of claim 1, wherein said4 dystrophin spectrin-like repeats are selected from the groupconsisting of: dystrophin spectrin-like repeat number 1, dystrophinspectrin-like repeat number 2, dystrophin spectrin-like repeat number 3,dystrophin spectrin-like repeat number 22, dystrophin spectrin-likerepeat number 23, and dystrophin spectrin like repeat number
 24. 34. Thecomposition of claim 16, wherein said 4 dystrophin spectrin-like repeatsare selected from the group consisting of: dystrophin spectrin-likerepeat number 1, dystrophin spectrin-like repeat number 2, dystrophinspectrin-like repeat number 3, dystrophin spectrin-like repeat number22, dystrophin speetrin-like repeat number 23, and dystrophin spectrinlike repeat number 24.